Activin a antibody formulations and methods of use thereof

ABSTRACT

The present invention provides novel pharmaceutical formulations comprising an antibody that specifically binds to human Activin A. The formulations may contain, in addition to an anti-Activin A antibody, a histidine buffer, an organic cosolvent, and a thermal stabilizer. The pharmaceutical formulations of the present invention exhibit a surprising degree of antibody stability after storage for several months and after being subjected to thermal and other physical stress.

RELATED APPLICATIONS

The instant application claims priority to U.S. Provisional ApplicationNo. 63/040,589, filed on Jun. 18, 2020, the entire contents of which areexpressly incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 30, 2021, isnamed 118003-75702_SL.txt and is 9,972 bytes in size.

FIELD

The present invention relates to the field of therapeutic antibodyformulations. More specifically, the present invention relates to thefield of pharmaceutical formulations comprising an antibody thatspecifically binds to human Activin A.

BACKGROUND

Fibrodysplasia ossificans progressiva (FOP), also known as Munchmeyerdisease, is an autosomal dominant disorder characterized by early onset,episodic and progressive ossification of skeletal muscle and associatedconnective tissue. In FOP subjects, bone forms in soft tissue outside ofthe normal skeleton, a process known as heterotopic ossification (HO),which can lead to the development of a secondary skeleton andprogressively restricts the patient's ability to move. Removal of thenew bone formation has been shown to be ineffective and leads to thedevelopment of additional new bone growth.

FOP is driven by mutations in the intracellular domain of ACVR1 (ALK2),with the great majority altering Arginine 206 to Histidine (R206H)(Pignolo, R. J. et al. 2011, Orphanet J. Rare Dis. 6:80). ACVR1 is atype I receptor for bone morphogenic proteins (BMPs). The R206Hmutation, among others, is believed to increase the sensitivity of thereceptor to activation and render it more resistant to silencing.

Although certain types of drugs have been used to relieve pain andswelling associated with FOP during flare-ups, no effective medicaltreatment is currently known for FOP. Antibodies to Activin A are oneexample of a therapeutically relevant macromolecule that requires properformulation. Although some anti-Activin A antibodies are known, therenonetheless remains a need in the art for novel pharmaceuticalformulations comprising anti-Activin A antibodies that are sufficientlystable and suitable for administration to patients.

SUMMARY

Methods to produce antibodies useful as human therapeutics includegeneration of chimeric antibodies and humanized antibodies (see, forexample, U.S. Pat. No. 6,949,245). See, for example, WO 94/02602(Abgenix) and U.S. Pat. No. 6,596,541 (Regeneron Pharmaceuticals), whichpublications are herein specifically incorporated by reference,describing methods of generating nonhuman transgenic mice capable ofproducing human antibodies. U.S. Pat. No. 9,718,881 discloses antibodiesto human Activin A, and is incorporated in its entirety herein byreference.

Therapeutic antibodies must be formulated in a manner that not onlymakes the antibodies suitable for administration to patients, but alsoin a manner that maintains their stability during storage and subsequentuse. For example, therapeutic antibodies in liquid solution are prone tofragmentation, precipitation, aggregation, and undesired chemicalmodifications unless the solution is formulated properly. The stabilityof an antibody in liquid formulation depends not only on the kinds ofexcipients used in the formulation, but also on the amounts andproportions of the excipients relative to one another. Furthermore,other considerations aside from stability must be taken into accountwhen preparing a liquid antibody formulation. Examples of suchadditional considerations include the viscosity of the solution and theconcentration of antibody that can be accommodated by a givenformulation, and the visual quality or appeal of the formulation. Thus,when formulating a therapeutic antibody, great care must be taken toarrive at a formulation that remains stable, contains an adequateconcentration of antibody, and possesses a suitable viscosity as well asother properties which enable the formulation to be convenientlyadministered to patients.

The present invention satisfies the aforementioned need by providingpharmaceutical formulations comprising a human antibody thatspecifically binds to human Activin A.

In one aspect, a liquid pharmaceutical formulation is provided,comprising: (i) an antibody that specifically binds to Activin A; (ii) abuffer; (iii) an organic cosolvent; and (iv) thermal stabilizers.

In another aspect, the present invention provides a pharmaceuticalformulation comprising: (i) an anti-human Activin A antibody, orantigen-binding portion thereof; (ii) a buffer at pH of 6.3±0.3; (iii)an organic cosolvent; and (iv) one or more thermal stabilizers.

In some embodiments, the antibody, or the antigen-binding portionthereof, comprises the following six CDR sequences: (a) an HCDR1 havingthe sequence GGSFSSHF (SEQ ID NO.: 1); (b) an HCDR2 having the sequenceILYTGGT (SEQ ID NO.: 2); (c) an HCDR3 having the sequenceARARSGITFTGIIVPGSFDI (SEQ ID NO.: 3); (d) an LCDR1 having the sequenceQSVSSSY (SEQ ID NO.: 4); (e) an LCDR2 having the sequence GAS (SEQ IDNO.: 5); and (f) an LCDR3 having the sequence QQYGSSPWT (SEQ ID NO.: 6).

In some embodiments, the antibody has a molecular weight of about145,235.3 Da.

In some embodiments, the concentration of the antibody, or theantigen-binding portion thereof, is 60 mg/mL±6 mg/mL.

In some embodiments, the buffer is a histidine buffer. In someembodiments, the histidine concentration is 10 mM±2 mM .

In some embodiments, the organic cosolvent is polysorbate 20. In someembodiments, the polysorbate 20 concentration is 0.05% w/v±0.025%.

In some embodiments, the one or more thermal stablilizers comprisesucrose and arginine. In some embodiments, the sucrose concentration is5%±1% (w/v) and the Arginine concentration is 70 mM±14 mM .

In some embodiments, the pharmaceutical formulation comprises 60 mg/mL±6mg/mL antibody, 10 mM±2 mM histidine, pH 6.3±0.3, 0.05% w/v±0.025%polysorbate 20, 5% w/v±1% sucrose, and 70 mM±14 mM Arginine.

In some embodiments, after 56 days of storage at 40° C. and 75% relativehumidity (RH), at least 90% of the antibody, or the antigen-bindingportion thereof, has native conformation , or at least 30% of theantibody, or the antigen-binding portion thereof, is the main chargeform.

In some embodiments, after 56 days of storage at 40° C. and 75% RH, atleast 93% of the antibody, or the antigen-binding portion thereof, hasnative conformation, or at least 34.5% of the antibody, or theantigen-binding portion thereof, is the main charge form.

In some embodiments, after 56 days of storage at 40° C. and 75% RH, atleast 97% of the antibody, or the antigen-binding portion thereof, hasnative conformation, or at least 45% of the antibody, or theantigen-binding portion thereof, is the main charge form.

In some embodiments, after six months of storage at 25° C. and 60% RH,at least 90% of the antibody, or the antigen-binding portion thereof,has native conformation, or at least 40% of the antibody, or theantigen-binding portion thereof, is the main charge form.

In some embodiments, after six months of storage at 25° C. and 60% RH,at least 95% of the antibody, or the antigen-binding portion thereof,has native conformation, or at least 45% of the antibody, or theantigen-binding portion thereof, is the main charge form.

In some embodiments, after six months of storage at 25° C. and 60% RH,at least 98% of the antibody, or the antigen-binding portion thereof,has native conformation, or at least 50% of the antibody, or theantigen-binding portion thereof, is the main charge form.

In some embodiments, after 12 months of storage at 2-8° C., at least 94%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 45% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 100% of the potency of the antibody , or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 12 months of storage at 2-8° C., at least 96%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 50% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 100% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 12 months of storage at 2-8° C., at least 98%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 55% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 100% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 18 months of storage at 2-8° C., at least 94%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 45% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 95% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 18 months of storage at 2-8° C., at least 96%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 50% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 95% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 18 months of storage at 2-8° C., at least 98%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 55% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 95% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 24 months of storage at 2-8° C., at least 94%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 45% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 95% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 24 months of storage at 2-8° C., at least 96%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 50% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 95% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In some embodiments, after 24 months of storage at 2-8° C., at least 98%of the antibody, or the antigen-binding portion thereof, has nativeconformation, at least 55% of the antibody, or the antigen-bindingportion thereof, is the main charge variant, and/or the antibody retainsat least 95% of the potency of the antibody, or the antigen-bindingportion thereof, prior to storage.

In another aspect, the present invention provides a pharmaceuticalformulation comprising (a) 60 mg/mL±10 mg/mL of an anti-human Activin Aantibody, or antigen-binding portion thereof (b) 10 mM±2 mM histidine,pH 6.3±0.3, (c) 0.05%±0.025% polysorbate 20, (d) 70 mM±14 mM Arginine,and (e) 5%±1% sucrose, wherein the antibody, or the antigen-bindingportion thereof, comprises a heavy chain variable region having at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% identity to,comprises, or consists of SEQ ID NO: 7 and a light chain variable regionhaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% identity to, comprises, or consists of SEQ ID NO: 8. In oneembodiment, the heavy chain comprises a sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% identity to,comprises, or consists of SEQ ID NO:9. In one embodiment, the heavychain comprises a sequence having at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% identity to, comprises, or consists ofSEQ ID NO:10.

In some embodiments, the formulation is contained in a container. Insome embodiments, the container is a vial. In some embodiments, the vialis glass. In some embodiments, the glass is Type 1 borosilicate glasswith a FluroTec® coated 4432/50 butyl rubber stopper.

In some embodiments, the formulation is suitable for intravenousadministration to a human subject in need thereof. In some embodiments,the formulation is suitable for subcutaneous administration to a humansubject in need thereof.

In some embodiments, the formulation is a liquid formulation. In someembodiments, the formulation is a lyophilized formulation.

In another aspect, the present invention provides a kit comprising apharmaceutical formulation disclosed herein, a container, andinstructions of use thereof.

In some embodiments, the container is a glass vial fitted with aFluroTec® coated chlorobutyl stopper.

In another aspect, the present invention provides a method of treating adisease or disorder associated with Activin A activity, the methodcomprising administration of a therapeutically effective amount of oneor more of the pharmaceutical compositions disclosed herein, to asubject in need thereof.

In some embodiments, the disease or disorder associated with Activin Aactivity is Fibrodysplasia ossificans progressiva (FOP).

Other embodiments of the present invention will become apparent from aeview of the ensuing detailed description.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the heavy and light chain amino acid sequences (SEQ ID NO:9and SEQ ID NO:10, respectively) of an exemplary anti-activin Amonoclonal antibody.

FIG. 2 shows the effect of varying the formulation on the rate of ananti-activin A antibody degradation when it is incubated at 40° C./75%RH for 2 months. For particle levels by HIAC and MFI the results shownare the final particle levels after 2 months of incubation (USP<788>limits are indicated for the HIAC results). Formulation 15 is thecontrol formulation in which all factors are nominal. The line is areference line indicating the rate of change of the response for thecontrol formulation.

FIG. 3 shows the effect of varying the formulation on the rate of ananti-activin A antibody degradation when it is incubated at 25° C./60%RH for 6 months. For particle levels by HIAC and MFI the results shownare the final particle levels after 6 months of incubation (USP<788>limits are indicated for the HIAC results). Formulation 15 is thecontrol formulation in which all factors are nominal. The line is areference line indicating the rate of change of the response for thecontrol formulation.

FIG. 4 shows the effects of varying the formulation components inaccordance with the DOE design on the stability of an anti-activin Aantibody relative to the control formulation, indicated as “deltacontrol” (8 cycles of freezing and thawing stress, −30° C. freeze, roomtemperature thaw; control formulation is F15). Results for particlelevels by HIAC are shown as final particle levels after 8 cycles offreezing and thawing and are compared to the limits specified inUSP<788>.

FIG. 5 shows the effects of varying the formulation components inaccordance with the DoE design on the stability of an anti-activin Aantibody relative to the control formulation, indicated as “deltacontrol” (120 minute agitation by vortexing; control formulation isF15). Results for particle levels by HIAC are shown as final particlelevels after 120 minutes of agitation and are compared to the limitsspecified in USP<788>.

FIG. 6 shows the effects of varying the formulation components inaccordance with the DOE design on the long-term stability of ananti-activin A antibody when the formulations were stored at 2-8° C. for12 months (see Table 24 for formulation descriptions). The limitsspecified in USP<788> are indicated on the plots showing the particlelevels by HIAC. Results are shown as a function of storage time at 2-8°C.

FIG. 7 shows the effects of varying the formulation components inaccordance with the DOE design on the long-term stability of ananti-activin A antibody when the formulations were stored at 2-8° C. for24 months (see Table 24 for formulation descriptions). The limitsspecified in USP<788> are indicated on the plots showing the particlelevels by HIAC. Results are shown as a function of storage time at 2-8°C.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about”, when used in reference to a particular recited numerical valueor range of values, means that the value may vary from the recited valueby no more than 5%. For example, as used herein, the expression “about100” includes 95 and 105 and all values in between (e.g., 95.00, 95.01,95.02, 95.03, 95.04, . . . , 104.96, 104.97, 104.98, 104.99, 105.00).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpublications mentioned herein are incorporated herein by reference intheir entirety.

Pharmaceutical Formulations

As used herein, the expression “pharmaceutical formulation” means acombination of at least one active ingredient (e.g., a small molecule,macromolecule, compound, etc. which is capable of exerting a biologicaleffect in a human or non-human animal), and at least one inactiveingredient which, when combined with the active ingredient or one ormore additional inactive ingredients, is suitable for therapeuticadministration to a human or non-human animal. The term “formulation”,as used herein, means “pharmaceutical formulation” unless specificallyindicated otherwise. The present invention provides pharmaceuticalformulations comprising at least one therapeutic polypeptide. Accordingto certain embodiments of the present invention, the therapeuticpolypeptide is an antibody, or an antigen-binding fragment thereof,which binds specifically to human Activin A protein. More specifically,the present invention includes pharmaceutical formulations thatcomprise: (i) a human antibody that specifically binds to human ActivinA (ii) a histidine buffer; (iii) an organic cosolvent that is anon-ionic surfactant; and (iv) a stabilizer that is a carbohydrate or aninorganic salt, or a combination of carbohydrate and inorganic salt.Specific exemplary components and formulations included within thepresent invention are described in detail below.

Anti-Activin A Antibodies

The pharmaceutical formulations of the present invention may comprise ahuman antibody, or an antigen-binding fragment thereof, that bindsspecifically to human Activin A. As used herein, the term “Activin A”means a human Activin A, which is a homo- or heterodimeric protein. Thehomodimeric protein contains a homodimeric beta A subunit pair. Theheterodimeric protein contains a beta subunit and a beta B, beta C orbeta E subunit (i.e., beta A beta B, beta A beta C, or beta A beta E.The subunits are each expressed as precursor polypeptides including asignal peptide, propeptide and mature polypeptide. An exemplary form ofhuman beta A subunit precursor is a polypeptide of length 426 aminoacids designated Swiss Prot P08476 of which residues 1-20 are a signalpeptide, residues 21-310 are a propeptide and residues 311-426 are themature polypeptide. An exemplary form of a beta B subunit precursorpolypeptide is designated Swiss Prot P09529 of which residues 1-28 are asignal peptide, residues 29-292 a propeptide and residues 293-407 amature polypeptide. An exemplary form of a beta C subunit is designatedSwiss Prot P55103, of which residues 1-18 are a signal peptide, residues19-236 are a propeptide and residues 237-352 are a mature polypeptide.An exemplary form of a beta E subunit precursor is designated Swiss ProtP58166 of which residues 1-19 are a signal peptide, residues 20-236 area propeptide and residues 237-350 are a mature polypeptide. Severalvariants of these sequences are known as described in the Swiss ProtData base. Reference to Activin A includes any of the beta A homodimer,beta A beta B, beta A beta C and beta A beta E heterodimer forms, aswell as their subunits, as well as their precursors in which subunitsare attached to the propeptide and/or signal peptide defined by theexemplary Swiss Prot sequences provided or other natural occurring humanforms of these sequences. Activin A signals through binding to ACVR2A orACVR2B, but is not known to be a ligand for ACVR1. Activin A signalsaberrantly via mutant ACVR to transduce osteogenic signals and triggerheterotopic bone formation.

The term “antibody”, as used herein, is generally intended to refer toimmunoglobulin molecules comprising four polypeptide chains: two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds,as well as multimers thereof (e.g., IgM); however, immunoglobulinmolecules consisting of only heavy chains (i.e., lacking light chains)are also encompassed within the definition of the term “antibody”. Eachheavy chain comprises a heavy chain variable region (abbreviated hereinas HCVR or V_(H)) and a heavy chain constant region. The heavy chainconstant region comprises three domains, CH1, CH2 and CH3. Each lightchain comprises a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region comprises one domain (CL1). The V_(H) and V_(L) regionscan be further subdivided into regions of hypervariability, termedcomplementary determining regions (CDRs), interspersed with regions thatare more conserved, termed framework regions (FR). Each V_(H) and V_(L)is composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

Unless specifically indicated otherwise, the term “antibody”, as usedherein, shall be understood to encompass complete antibody molecules aswell as antigen-binding fragments thereof. The term “antigen-bindingportion” or “antigen-binding fragment” of an antibody (or simply“antibody portion” or “antibody fragment”), as used herein, refers toone or more fragments of an antibody that retain the ability tospecifically bind to human Activin A or an epitope thereof.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds human Activin A is substantially free of antibodies thatspecifically bind antigens other than human Activin A), with the notableexception of bi-specific (or multi-specific) antibodies thatspecifically bind Activin A on the one hand, and another epitope on theother. Moreover, an isolated antibody may be substantially free of othercellular material or chemicals.

The term “specifically binds”, or the like, means that an antibody orantigen-binding fragment thereof forms a complex with an antigen that isrelatively stable under physiologic conditions. Specific binding can becharacterized by a dissociation constant of at least about 1×10⁻⁶ M orgreater. Methods for determining whether two molecules specifically bindare well known in the art and include, for example, equilibriumdialysis, surface plasmon resonance, and the like. An isolated antibodythat specifically binds human Activin A may, however, havecross-reactivity to other antigens, such as Activin A molecules fromother species (orthologs). In the context of the present invention,multispecific (e.g., bispecific) antibodies that bind to human Activin Aas well as one or more additional antigens are deemed to “specificallybind” human Activin A.

Exemplary anti-human Activin A antibodies that may be included in thepharmaceutical formulations of the present invention are set forth inU.S. Pat. No. 9,718,881, the disclosure of which is incorporated hereinby reference in their entirety.

Antibodies can also include antibodies specifically binding to ActivinA. Such antibodies can specifically bind to any or all of the beta Abeta A, beta A beta B, beta A beta C and beta A beta E forms of ActivinA. Some antibodies specifically bind to only one of these forms (i.e.,beta A beta A, beta A beta B, beta A beta C or beta A beta E).Specificity for the beta A beta B, beta A beta C and beta A beta E formscan be conferred by an epitope within the beta B, beta C or beta Esubunit, respectively, or for an epitope to which both components of theheterodimer contribute. Specificity for beta A beta can be conferred byan epitope contributed by both molecules within the homodimer (e.g., atthe interface of subunits). Some antibodies specifically bind to all ofthese forms of Activin A, in which case the epitope is typically on thebeta A subunit. Antibodies typically have epitopes within the maturepolypeptide component of the precursor proteins. Some antibodiesspecifically bind to any or all forms of Activin A without binding tohuman inhibin, which exists in the form of alpha (Swiss Prot P05111)beta A or alpha beta B heterodimers. Some antibodies specifically bindto any or all forms of Activin A and bind to either or both forms ofhuman inhibin. Although it is believed that such antibodies inhibitsignal transduction of Activin A through one or more of itscounterreceptors, ACVR2A and/or ACVR2B and/or BMPR2, an understanding ofmechanism is not required for use of such antibodies in methods oftreating FOP.

A substantial number of antibodies against Activin A have beendescribed. For example, US20150037339 discloses human antibodiesdesignated H4H10423P, H4H10424P, H4H10426P, H4H10429P, H4H10430P,H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2, H4H10438P2, H4H10440P2,H4H10442P2, H4H10445P2, H4H10446P2, H4H10447P2, H4H10447P2, H4H10448P2,H4H10452P2.

Preferred antibodies have an affinity for Activin A (measured at 25° C.as in Example 3 of U.S. Pat. No. 9,718,881) of at least 10⁸ M⁻¹, 10⁹M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, or 10¹³ M⁻¹. Some antibodies have anaffinity within a range of 10⁹-10¹² M⁻¹. Preferred antibodies inhibitsignal transduction of Activin A with an IC50 of less than 4 nM, andpreferably less than 400 pM or 40 pM. Some antibodies inhibit signaltransduction with and IC50 in a range of 4 nM to 10 pM or 3.5 nM to 35pM.

Signal transduction inhibition can be measured as in Example 6 of U.S.Pat. No. 9,718,881, which is summarized as follows. A human A204rhabdomyosarcoma cell line is transfected with a Smad 2/3-luciferasereporter plasmid to produce the A204/CAGAx12-Luc cell line.A204/CAGAx12-Luc cells were maintained in McCoy's 5A supplemented with10% fetal bovine serum, penicillin/streptomycin/glutamine and 250 μg/mLof G418. For the bioassay, A204/CAGAx12-Luc cells were seeded onto96-well assay plates at 10,000 cells/well in low serum media, 0.5% FBSand OPTIMEM, and incubated at 37° C. and 5% CO₂ overnight. Activin A isserially diluted at 1:3 from 100 to 0.002 nM and added to cells startingalong with a control containing no Activin. Antibodies are seriallydiluted at 1:3 starting from 100 to 0.002 nM, 1000 to 0.02 nM, or 300 to0.005 nM including control samples containing either an appropriateisotype control antibody or no antibody and added to cells with aconstant concentration of 100 pM Activin A.

Some antibodies inhibit binding of Activin A to ACVR2A and/or ACVR2Band/or BMPR2 by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, as measured when the receptor is expressed from a cell orthe extracellular domain is fused with an Fc domain as a fusion protein,and the fusion protein is immobilized to support (e.g., a Biacore sensorchip). In such measurements, the antibody and Activin A should bepresent in equimolar amounts and the receptor or extracellular domain inexcess.

An exemplary antibody used in the present examples is designatedH4H10446P in U.S. Pat. No. 9,718,881. Its heavy chain variable regionand heavy chain CDR1, CDR2 and CDR3 having the amino acid sequences ofSEQ ID NOs:162, 164, 166 and 168, respectively, of U.S. Pat. No.9,718,881. Its light chain variable region and light chain CDRs, CDRL1,CDRL2 and CDRL3 having the amino acid sequences of SEQ ID NO:146, 148,150 and 152, respectively, of US2015/0037339. H4H10446P inhibits ActivinA mediated signaling through ACVR2A and/or ACVRIIB, but does not inhibitstrongly, if at all, Activin A binding to ACRIIA or ACVR2B. Otherantibodies competing with H4H10446P for binding to human Activin A orbinding to the same epitope on human Activin A as H4H10446P are includedand sharing its inhibition of signaling are also included.

Another exemplary antibody for use in the present methods is designatedH4H10430P in U.S. Pat. No. 9,718,881. Its heavy chain variable regionand heavy chain CDRs CDRH1, CDRH2 and CDRH3 having the amino acidsequences of SEQ ID NOs: 66, 68, 70 and 72, respectively, in U.S. Pat.No. 9,718,881. Its light chain variable region and light chain CDRs,CDRL1, CDRL2 and CDRL3 having the amino acid sequences of SEQ ID NOs:74,76, 78 and 80, respectively, in U.S. Pat. No. 9,718,881. This antibodyinhibits binding of Activin A to ACRV2A and/or ACVR2B and inhibitssignal transduction through one or both of these receptors. Otherantibodies competing with H4H10430P for binding to Activin A or bindingto the same epitope on Activin A as H4H10430P and sharing its propertyof inhibiting Activin A binding to and signal transduction throughACVR2A and ACVR2B are also included.

An exemplary antibody for use in the present methods is garetosmab. Therecombinant monoclonal antibody garetosmab is a covalent heterotetramerconsisting of two disulfide-linked human heavy chains (IgG4 isotype),each covalently linked through a disulfide bond to a human kappa lightchain. Based on the primary sequence, the antibody without glycanspossesses a predicted molecular weight of 145,235.3 Da, assuming theformation of 16 canonical disulfide bonds and removal of Lys453 fromeach heavy chain C-terminus. Each heavy chain contains aserine-to-proline mutation at amino acid Pro234 within the hinge regionof the Fc domain, to reduce the propensity of the IgG4 isotype antibodyto form half-antibodies in solution. There is a single N-linkedglycosylation site (Asn303) on each heavy chain, located within theconstant region in the Fc domain of the molecule. Thecomplementarity-determining regions (CDRs) within the garetosmab heavychain and light chain variable domains together form the binding sitefor its targets: activin A, activin AB, and activin AC. The heavy andlight chain amino acid sequences, the location of the CDRs within eachpolypeptide chain, the location of the heavy chain N-linkedglycosylation site, and the predicted disulfide bond structures of thegaretosmab monoclonal antibody are presented in FIG. 1.

In some embodiments, the anti-human Activin A antibody comprises one ormore amino acid substitutions in one or more framework regions relativeto the canonical heavy chain variable region, which is reasonablyexpected to result in an altered charge distribution across the exposedsurface of the antibody, and therefore affect its interaction with thesurrounding solvent and excipients.

The amount of antibody, or antigen-binding fragment thereof, containedwithin the pharmaceutical formulations of the present invention may varydepending on the specific properties desired of the formulations, aswell as the particular circumstances and purposes for which theformulations are intended to be used. In certain embodiments, thepharmaceutical formulations are liquid formulations that may contain 20mg/mL±2 mg/mL to 200 mg/mL±20 mg/mL of antibody; 30±3 mg/mL to 150±15mg/mL of antibody; 40±4 mg/mL to 100±1 mg/mL of antibody; 45±4.5 mg/mLto 80±8 mg/mL of antibody; 50±5 mg/mL to 60±6 mg/mL of antibody; 55±5.5mg/mL to 60±6 mg/mL of antibody; about 50 mg/mL; 50 mg/mL; 60 mg/mL±6mg/mL; about 60 mg/mL; 60 mg/mL; about 70 mg/mL; or 70 mg/mL of anantibody or an antigen-binding fragment thereof, that binds specificallyto human Activin A.

Excipients and pH

The pharmaceutical formulations of the present invention comprise one ormore excipients. The term “excipient”, as used herein, means anynon-therapeutic agent added to the formulation to provide a desiredconsistency, viscosity or stabilizing effect.

In certain embodiments, the pharmaceutical formulation of the inventioncomprises at least one organic cosolvent in a type and in an amount thatstabilizes the human Activin A antibody under stress conditions of roughhandling or agitation, such as, e.g., vortexing, or freezing andthawing. In some embodiments, what is meant by “stabilizes” is themaintenance of at least 95% of the Activin A antibody in its nativestate, i.e., not fragmented or aggregated, of antibody (on a molarbasis) over the course of rough handling, such as by vortexing theantibody-organic cosolvent solution for about 30 minutes, 60 minutes orabout 120 minutes, or by freezing and thawing the antibody-organiccosolvent solution for 4 cycles or 8 cycles.

In certain embodiments, the organic cosolvent is a non-ionic surfactant,such as an alkyl poly(ethylene oxide). Specific non-ionic surfactantsthat can be included in the formulations of the present inventioninclude, e.g., polysorbates such as polysorbate 20, polysorbate 28,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80,polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 181,poloxamer 188, poloxamer 407; or polyethylene glycol (PEG). Polysorbate20 is also known as TWEEN 20, sorbitan monolaurate andpolyoxyethylenesorbitan monolaurate. Poloxamer 188 is also known asPLURONIC F68.

The amount of non-ionic surfactant contained within the pharmaceuticalformulations of the present invention may vary depending on the specificproperties desired of the formulations, as well as the particularcircumstances and purposes for which the formulations are intended. Incertain embodiments, the formulations may contain 0.01%±0.0015% to1%±0.15% surfactant. For example, the formulations of the presentinvention may comprise about 0.0085%; about 0.01%; about 0.02%; about0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%;about 0.09%; about 0.1%; about 0.11%; about 0.12%; about 0.13%; about0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%;about 0.20%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about0.25%; about 0.3%; about 0.4%; about 0.5%; about 0.6%; about 0.7%; about0.8%; about 0.9%; about 1%; about 1.1%; about 1.15%; or about 1.2%polysorbate 20.

The pharmaceutical formulations of the present invention may alsocomprise one or more stabilizers in a type and in an amount thatstabilizes the human Activin A antibody under conditions of thermalstress. In some embodiments, what is meant by “stabilizes” ismaintaining greater than about 90% of the antibody in a nativeconformation when the solution containing the antibody and the thermalstabilizer is kept at about 2-8° C. for up to about 18 months, at about25° C. for up to about 6 months, or at about 40° C. for up to about 56days. In some embodiments, what is meant by “stabilizes” is maintaininggreater than about 90% of the antibody in a native conformation when thesolution containing the antibody, or antigen-binding fragment thereof,and the thermal stabilizer is kept at about 2-8° C. for up to about 24months. As used herein, “native” means the major form of the antibody bysize exclusion, which is generally an intact monomer of the antibody.

In certain embodiments, the thermal stabilizer is a sugar or sugaralcohol selected from sucrose, sorbitol, glycerol, trehalose andmannitol, or any combination thereof, the amount of which containedwithin the formulation can vary depending on the specific circumstancesand intended purposes for which the formulation is used. In certainembodiments, the formulations may contain about 5% to about 40% sugar orsugar alcohol; about 1% to about 20% sugar or sugar alcohol; about 5% toabout 15% sugar or sugar alcohol; about 7.5% to about 12.5% sugar orsugar alcohol; about 10% sugar or sugar alcohol; 10%±1.5% sugar or sugaralcohol; or 10% sugar or sugar alcohol. For example, the pharmaceuticalformulations of the present invention may comprise 1%±0.2%; 2%±0.4%;3%±0.6%; 4%±0.8%; 5%±1%; 6%±1.2%; 7%±1.4%; 8%±1.6%; 9%±1.8%; 10%±2%;11%±2.2%; 12%±2.4%; 13%±2.6%; 14%±2.8%; or about 15%±3% (w/v) of sugaror sugar alcohol (e.g., sucrose).

In certain embodiments, the thermal stabilizer is an amino acid (e.g.,arginine), the amount of which contained within the formulation can varydepending on the specific circumstances and intended purposes for whichthe formulation is used. In certain embodiments, the formulations maycontain about 0 mM to about 150 mM arginine; about 10 mM to about 140 mMarginine; about 20 mM to about 130 mM arginine; about 30 mM to about 120mM arginine; about 40 mM to about 110 mM arginine; about 50 mM to about100 mM arginine; about 60 mM to about 90 mM arginine; about 70 mM toabout 80 mM arginine; or about 70 mM arginine. For example, thepharmaceutical formulations of the present invention may comprise 0 mM;10 mM±2 mM arginine; 20 mM±4 mM arginine; 30 mM±6 mM arginine; 40 mM±8mM arginine; 50 mM±10 mM arginine; 60 mM±12 mM arginine; 70 mM±14 mMarginine; about 70 mM arginine; or 70 mM arginine.

The pharmaceutical formulations of the present invention may alsocomprise a buffer or buffer system, which serves to maintain a stable pHand to help stabilize the human Activin A antibody. In some embodiments,what is meant by “stabilizes” is wherein at least 90% of the antibody isin its native conformation as determined by size exclusionchromatography when the solution containing the antibody and the bufferis kept at about 2-8° C. for up to about 18 months, at about 25° C. forup to about 6 months, or at about 40° C. for up to about 56 days. Insome embodiments, what is meant by “stabilizes” is wherein at least 90%of the antibody is in its native conformation as determined by sizeexclusion chromatography when the solution containing the antibody, orthe antigen-binding fragment thereof, and the buffer is kept at about2-8° C. for up to about 24 months. By “native” or “native conformation”,what is meant is the antibody fraction that is not aggregated ordegraded. This is generally determined by an assay that measures therelative size of the antibody entity, such as a size exclusionchromatographic assay. The non-aggregated and non-fragmented antibodyelutes at a fraction that equates to the native antibody, and isgenerally the main elution fraction. Aggregated antibody elutes at afraction that indicates a size greater than the native antibody.Fragmented antibody elutes at a fraction that indicates a size less thanthe native antibody.

In some embodiments, what is meant by “stabilizes” is wherein at least40% of the antibody is in its main charge form as determined by cationexchange chromatography when the solution containing the antibody andthe buffer is kept at about 2-8° C. for up to about 18 months, at about25° C. for up to about 6 months, or at about 40° C. for up to about 56days. In some embodiments, what is meant by “stabilizes” is wherein atleast 40% of the antibody is in its main charge form as determined bycation exchange chromatography when the solution containing theantibody, or the antigen-binding fragment thereof, and the buffer iskept at about 2-8° C. for up to about 24 months. By “main charge” or“main charge form”, what is meant is the fraction of antibody thatelutes from an ion exchange resin in the main peak, which is generallyflanked by more “basic” peaks on one side and more “acidic” peaks on theother side.

The pharmaceutical formulations of the present invention may have a pHof from about 4.5 to about 7.0. For example, the formulations of thepresent invention may have a pH of about 4.5; about 4.6; about 4.7;about 4.8; about 4.9; about 5.0; about 5.1; about 5.2; about 5.3; about5.4; about 5.5; about 5.6; about 5.7; about 5.8; about 5.9; about 6.0;about 6.1; about 6.2; about 6.3; about 6.4; about 6.5; or about 6.6. Insome embodiments, the pH is 6.3±0.5; 6.3±0.4; 6.3±0.3; 6.3±0.2; 6.3±0.1;about 6.3; or 6.3.

In some embodiments, the buffer or buffer system comprises at least onebuffer that has a buffering range that overlaps fully or in part therange of pH 5.5-7.4, such as, e.g., a buffer having a useful bufferingrange of pH 4.8 to pH 8.8. In one embodiment, the buffer has a pH ofabout 6.3. In certain embodiments, the buffer comprises a histidinebuffer. In certain embodiments, the histidine is present at aconcentration of 5 mM±1 mM to 25 mM±5 mM; 6 mM±1.2 mM to 20 mM±4 mM; 7mM±1.4 mM to 15 mM±3 mM; 8 mM±1.6 mM to 12 mM±2.4 mM; 9 mM±1.8 mM to 11mM±2.2 mM; 10 mM±2 mM; about 10 mM; or 10 mM. In certain embodiments,the buffer system comprises phosphate at 10 mM±2 mM, at a pH of 6.3±0.3.

Exemplary Formulations

According to one aspect of the present invention, the liquidpharmaceutical formulation comprises: (i) 60 mg/mL±6 mg/mL of a humanantibody that specifically binds to human Activin A (e.g., garetosmab);(ii) a buffer system that buffers at about pH 6.3±0.3; (iii) a thermalstabilizer comprising a sugar and a salt; and (iv) an organic cosolvent.

According to one embodiment, the pharmaceutical formulation comprises:(i) 20±2 mg/mL to 200±20 mg/mL human antibody that specifically binds tohuman Activin A; (ii) a histidine buffer, which buffers at pH 6.3±0.3;(iii) sucrose and arginine; and (iv) a non-ionic detergent, such as apolysorbate.

According to one embodiment, the pharmaceutical formulation comprises:(i) 50 mg/ml±5 mg/mL human IgG1 antibody that specifically binds tohuman Activin A, and which comprises an HCDR1 of SEQ ID NO: 1, an HCDR2of SEQ ID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, anLCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6; (ii) 10 mM±2 mMhistidine, pH 6.3±0.3; (iii) 5%±1% sucrose; (iv) 70 mM ±14 mM arginine;and (v) 0.05%±0.025% polysorbate 20.

According to one embodiment, the pharmaceutical formulation comprises:(i) 60 mg/ml±6 mg/mL human antibody that specifically binds to humanActivin A, and which comprises an HCDR1 of SEQ ID NO: 1, an HCDR2 of SEQID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, an LCDR2of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6; (ii) 10 mM±2 mMhistidine, pH 6.3±0.3; (iii) 5%±1% sucrose; (iv) 70 mM±14 mM arginine;and (v) 0.05%±0.025% polysorbate 20.

According to one embodiment, the pharmaceutical formulation comprises:(i) 70 mg/ml±7 mg/mL human IgG1 antibody that specifically binds tohuman Activin A, and which comprises an HCDR1 of SEQ ID NO: 1, an HCDR2of SEQ ID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, anLCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6, at a concentrationof; (ii) 10 mM±2 mM histidine pH 6.3±0.3; (iii) 5%±1% sucrose; (iv) 70mM±14 mM arginine; and (iv) 0.05%±0.025% polysorbate 20.

According to one embodiment, the pharmaceutical formulation comprises:(i) 60 mg/ml ±6 mg/mL human antibody that specifically binds to humanActivin A, and which comprises a heavy chain variable domain of SEQ IDNO: 1, and a light chain variable domain of SEQ ID NO: 5; (ii) 10 mM±2mM histidine, pH 6.3±0.3; (iii) 5%±1% sucrose; (iv) 70 mM ±14 mMarginine; and (v) 0.05%±0.025% polysorbate 20.

Exemplary formulations and primary container closures used duringgaretosmab preclinical and clinical development are listed in Table.

TABLE 1 Garetosmab Drug Product Formulations Tested During InitialToxicology Studies, Phase 1, and Phase 2 Clinical Studies ToxicologyPhase 1 Phase 2/Commercial Garetosmab 50 mg/mL 50 mg/mL 60 mg/mLconcentration L-histidine 10 mM 10 mM 10 mM pH 6.3 6.3 6.3 Sucrose   5%(w/v)   5% (w/v)   5% (w/v) L-arginine HCl — — 70 mM Polysorbate 80 0.1%(w/v) 0.1% (w/v) — Polysorbate 20 — — 0.05% (w/v) Drug Product FormFrozen Liquid Lyophilized Liquid Primary Type I Glass vial with Type IGlass vial with Type I Glass vial with Container/Closure FluroTec ®coated FluroTec ® coated FluroTec ® coated chlorobutyl stopperchlorobutyl stopper chlorobutyl stopper

Additional non-limiting examples of pharmaceutical formulationsencompassed by the present invention are set forth elsewhere herein,including the working Examples presented below.

Stability and Robustness of the Pharmaceutical Formulations

The pharmaceutical formulations of the present invention typicallyexhibit high levels of stability. The term “stable”, as used herein inreference to the pharmaceutical formulations, means that the antibodieswithin the pharmaceutical formulations retain an acceptable degree ofphysical and/or chemical structure or biological function after storageunder defined conditions. A formulation may be stable even though theantibody contained therein does not maintain 100% of its physical and/orchemical structure or biological function after storage for a definedamount of time. Under certain circumstances, maintenance of at leastabout 90%, 95%, 96%, 97%, 98% or 99% of an antibody's structure orfunction after storage for a defined amount of time may be regarded as“stable”.

Stability can be measured, inter alia, by determining the percentage ofnative antibody that remains in the formulation after storage for adefined amount of time at a defined temperature and/or relative humidity(RH). The percentage of native antibody can be determined by, interalia, size exclusion chromatography (e.g., size exclusion ultraperformance liquid chromatography [SE-UPLC]), reversed-phasechromatography (e.g., reversed-phase ultra performance liquidchromatography [RP-UPLC]), and/or microchip capillary electrophoresis(e.g., reduced MCE or non-reduced MCE), such that native meansnon-aggregated and non-fragmented. An “acceptable degree of stability”,as that phrase is used herein, means that at least 90% of the nativeform of the antibody can be detected in the formulation after storagefor a defined amount of time at a given temperature. In certainembodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% of the native form of the antibody can be detected in theformulation after storage for a defined amount of time at a definedtemperature. The defined amount of time after which stability ismeasured can be at least 7 days, at least 14 days, at least 21 days, atleast 28 days, at least 56 days, at least 1 month, at least 2 months, atleast 3 months, at least 4 months, at least 5 months, at least 6 months,at least 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, at least 12 months, at least 18 months, atleast 24 months, or more. The defined temperature at which thepharmaceutical formulation may be stored when assessing stability can beany temperature from about −80° C. to about 45° C., e.g., storage atabout −80° C., about −30° C., about −20° C., about 0° C., about 2° -8°C., about 5° C., about 25° C., about 40° C., or about 45° C. The definedrelative humidity (RH) at which the pharmaceutical formulation may bestored when assessing stability can be about 20-90% RH, about 20% RH,about 25% RH, about 30% RH, about 35% RH, about 40% RH, about 45% RH,about 50% RH, about 55% RH, about 60% RH, about 65% RH, about 70% RH,about 75% RH, about 80% RH, about 85% RH, or about 90% RH. Stressconditions may be applied to the pharmaceutical formulation to assessstability, e.g., vortexing, or freezing and thawing. Stress conditionsinclude, for example, vortexing the antibody-organic cosolvent solutionfor about 30 minutes, 60 minutes or about 120 minutes, or freezing andthawing the antibody-organic cosolvent solution for 4 cycles or 8cycles. Thermal stress may be applied to the pharmaceutical formulationto assess stability. Thermal stress includes for example holding theformulation for about 14 days, about 28 days or about 56 days at about25° C., about 35° C., about 37° C., about 40° C. or about 45° C. Incertain embodiments, stress may be applied to the pharmaceuticalformulation for about 56 days at about 40° C. and about 75% RH.

For example, a pharmaceutical formulation may be deemed stable if aftertwenty-four months of storage at 2-8° C., at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% of antibody, orantigen-binding fragment thereof, detected by SE-UPLC, RP-UPLC or MCE isnative (i.e., in the native peak fraction). A pharmaceutical formulationmay be deemed stable if after eighteen months of storage at 2-8° C., atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%of antibody detected by SE-UPLC, RP-UPLC or MCE is native (i.e., in thenative peak fraction). A pharmaceutical formulation may be deemed stableif after six months of storage at 25° C., at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% of antibody detected bySE-UPLC, RP-UPLC or MCE is native (i.e., in the native peak fraction). Apharmaceutical formulation may be deemed stable if after six months ofstorage at 25° C. and at 60% RH, at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 98.5%, 99% or 99.5% of antibody detected by SE-UPLC,RP-UPLC or MCE is native (i.e., in the native peak fraction). Apharmaceutical formulation may also be deemed stable if after six monthsof storage at 25° C. at least 97%, 97.5%, 98%, 98.5%, 99% or 99.5% ofantibody detected by SE-UPLC, RP-UPLC or MCE is native. A pharmaceuticalformulation may also be deemed stable if after six months of storage at25° C. and at 60% RH, at least 97%, 97.5%, 98%, 98.5%, 99% or 99.5% ofantibody detected by SE-UPLC, RP-UPLC or MCE is native. A pharmaceuticalformulation may be deemed stable if after three months of storage at 25°C., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or99.5% of antibody detected by SE-UPLC, RP-UPLC or MCE is native (i.e.,in the native peak fraction). A pharmaceutical formulation may be deemedstable if after one month of storage at 25° C., at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5% of antibody detectedby SE-UPLC, RP-UPLC or MCE is native (i.e., in the native peakfraction). A pharmaceutical formulation may also be deemed stable ifafter 56 days of storage at 40° C., at least 96%, 96.5%, 97%, 97.5%,98%, 98.5%, 99% or 99.5% of antibody detected by SE-UPLC, RP-UPLC or MCEis native. A pharmaceutical formulation may also be deemed stable ifafter 56 days of storage at 40° C. and at 75% RH, at least 96%, 96.5%,97%, 97.5%, 98%, 98.5%, 99% or 99.5% of antibody detected by SE-UPLC,RP-UPLC or MCE is native. A pharmaceutical formulation may also bedeemed stable if after 56 days of storage at 40° C. at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of antibody detected bySE-UPLC, RP-UPLC or MCE is native. A pharmaceutical formulation may alsobe deemed stable if after 56 days of storage at 40° C. and at 75% RH, atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of antibodydetected by SE-UPLC, RP-UPLC or MCE is native. A pharmaceuticalformulation may also be deemed stable if after six months of storage at−20° C. at least 98.5%, 99% or 99.5% of antibody detected by SE-UPLC,RP-UPLC or MCE is native. A pharmaceutical formulation may also bedeemed stable if after 18 months of storage at −30° C. at least 99% or99.5% of antibody detected by SE-UPLC, RP-UPLC or MCE is native. Apharmaceutical formulation may also be deemed stable if after six monthsof storage at −80° C. at least 99% or 99.5% of antibody detected bySE-UPLC is native.

Stability can be measured, inter alia, by determining the percentage ofantibody that migrates in the main fraction of antibody (“main chargeform”) during ion exchange, relative to the total combined peak area,wherein stability is proportional to the fraction of antibody in themain charge form. The charge form can be determined by, inter alia,cation exchange chromatography (e.g., cation exchange ultra performanceliquid chromatography [CEX-UPLC]), or imaged capillary isoelectricfocusing (iCIEF). While not wishing to be bound by theory, deamidationof the antibody may cause the antibody to become more negatively chargedand thus more acidic relative to the non-deamidated antibody (see, e.g.,Robinson, N., Protein Deamidation, PNAS, Apr. 16, 2002,99(8):5283-5288). An “acceptable degree of stability”, as that phrase isused herein, means that at least 30% of the antibody is in a main chargeform detected in the formulation after storage for a defined amount oftime at a defined temperature. In certain embodiments an acceptabledegree of stability means that at least about 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the antibody can bedetected in a main charge form after storage for a defined amount oftime at a given temperature, or under thermal, freeze/thaw, or agitationstress. The defined amount of time after which stability is measured canbe at least 7 days, at least 14 days, at least 28 days, at least 1month, at least 2 months, at least 3 months, at least 4 months, at least5 months, at least 6 months, at least 7 months, at least 8 months, atleast 9 months, at least 10 months, at least 11 months, at least 12months, at least 18 months, at least 24 months, or more. The temperatureat which the pharmaceutical formulation may be stored when assessingstability can be any temperature from about −80° C. to about 45° C.,e.g., storage at about −80° C., about −30° C., about −20° C., about 0°C., about 2° -8° C., about 5° C., about 25° C., about 37° C., about 40°C. or about 45° C. The relative humidity (RH) at which thepharmaceutical formulation may be stored when assessing stability can beabout 20-90% RH, about 20% RH, about 25% RH, about 30% RH, about 35% RH,about 40% RH, about 45% RH, about 50% RH, about 55% RH, about 60% RH,about 65% RH, about 70% RH, about 75% RH, about 80% RH, about 85% RH, orabout 90% RH. For example, a pharmaceutical formulation may be deemedstable if after 24 months of storage at 2-8° C. no less than about 30%,35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or61% of the antibody, or antigen-binding fragment thereof, is in a maincharge form. A pharmaceutical formulation may be deemed stable if after18 months of storage at 2-8° C. no less than about 30%, 35%, 40%, 45%,50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or 61% of theantibody is in a main charge form. A pharmaceutical formulation may alsobe deemed stable if after six months of storage at 25° C. and 60% RH, noless than about 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, or 61% of the antibody is in a main charge form. Apharmaceutical formulation may also be deemed stable if after six monthsof storage at 25° C., no less than about 45% of the antibody is in amain charge form. A pharmaceutical formulation may also be deemed stableif after 56 days of storage at 40° C. and 75% RH, no less than about30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, or 61% of the antibody is in a main charge form. A pharmaceuticalformulation may also be deemed stable if after 56 days of storage at 40°C., no less than about 34% of the antibody can be detected in a maincharge form.

Measuring the binding affinity of the antibody to its target may also beused to assess stability or potency. The term “potency” refers to any ofthe factors that contribute to the utility or biological activity of anantibody to recognize an antigen. Potency can be assayed byligand-binding assays or functional assays, for example, ELISA, flowcytometry and/or other in vitro cell-based assays. For example, aformulation of the present invention may be regarded as stable if, afterstorage at e.g., −80° C., −30° C., −20° C., 5° C., 25° C., 37° C., 40°C., 45° C., etc. for a defined amount of time (e.g., 7 days to 24months), the anti-Activin A antibody contained within the formulationmaintains at least 50% and up to about 150% of the potency, measured asbinding affinity, of the antibody prior to said storage. Bindingaffinity may be determined by e.g., ELISA or plasmon resonance.Biological activity may be determined by a Activin A activity assay,such as e.g., by contacting a cell that expresses Activin A with theformulation comprising the anti Activin A antibody. The binding of theantibody to such a cell may be measured directly, such as via FACSanalysis. Alternatively, the downstream activity of the Activin Asignaling pathway may be measured in the presence of the antibody, andcompared to the activity of the Activin A signaling pathway in theabsence of antibody. In some embodiments, the Activin A may beendogenous to the cell. In other embodiments, the Activin A may beectopically (heterologously) expressed in the cell.

Additional methods for assessing the stability of an antibody informulation are demonstrated in the Examples presented below.

Containers and Methods of Administration

The pharmaceutical formulations of the present invention may becontained within any container suitable for storage or administration ofmedicines and other therapeutic compositions. For example, thepharmaceutical formulations may be contained within a sealed andsterilized plastic or glass container having a defined volume such as avial, ampule, syringe, cartridge, bottle, or IV bag. Different types ofvials can be used to contain the formulations of the present inventionincluding, e.g., clear and opaque (e.g., amber) glass or plastic vials.Likewise, any type of syringe can be used to contain or administer thepharmaceutical formulations of the present invention.

The pharmaceutical formulations of the present invention may becontained within “normal tungsten” syringes or “low tungsten” syringes.As will be appreciated by persons of ordinary skill in the art, theprocess of making glass syringes generally involves the use of a hottungsten rod which functions to pierce the glass thereby creating a holefrom which liquids can be drawn and expelled from the syringe. Thisprocess results in the deposition of trace amounts of tungsten on theinterior surface of the syringe. Subsequent washing and other processingsteps can be used to reduce the amount of tungsten in the syringe. Asused herein, the term “normal tungsten” means that the syringe containsgreater than or equal to 500 parts per billion (ppb) of tungsten. Theterm “low tungsten” means that the syringe contains less than 500 ppb oftungsten. For example, a low tungsten syringe, according to the presentinvention, can contain less than about 490, 480, 470, 460, 450, 440,430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50,40, 30, 20, 10 or fewer ppb of tungsten.

The rubber plungers used in syringes, and the rubber stoppers used toclose the openings of vials, may be coated to prevent contamination ofthe medicinal contents of the syringe or vial, or to preserve theirstability. Thus, pharmaceutical formulations of the present invention,according to certain embodiments, may be contained within a syringe thatcomprises a coated plunger, or within a vial that is sealed with acoated rubber stopper. For example, the plunger or stopper may be coatedwith a fluorocarbon film. Examples of coated stoppers or plungerssuitable for use with vials and syringes containing the pharmaceuticalformulations of the present invention are mentioned in, e.g., U.S. Pat.Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, thecontents of which are incorporated by reference herein in theirentireties. Particular exemplary coated rubber stoppers and plungersthat can be used in the context of the present invention arecommercially available under the tradename “FluroTec®”, available fromWest Pharmaceutical Services, Inc. (Lionville, Pa.). FluroTec® is anexample of a flurocarbon coating used to minimize or prevent drugproduct from adhering to the rubber surfaces.

According to certain embodiments of the present invention, thepharmaceutical formulations may be contained within a low tungstensyringe that comprises a fluorocarbon-coated plunger.

The pharmaceutical formulations can be administered to a patient byparenteral routes such as injection (e.g., subcutaneous, intravenous,intramuscular, intraperitoneal, etc.) or percutaneous, mucosal, nasal,pulmonary or oral administration. Numerous reusable pen or autoinjectordelivery devices can be used to subcutaneously deliver thepharmaceutical formulations of the present invention. Examples include,but are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland),HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly andCo., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk,Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen,Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPEN™,OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,Frankfurt, Germany). Examples of disposable pen or autoinjector deliverydevices having applications in subcutaneous delivery of a pharmaceuticalcomposition of the present invention include, but are not limited to theSOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and theKWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks,Calif.), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey,L.P.), and the HUMIRA™ Pen (Abbott Labs, Abbott Park, Ill.).

The use of a microinfusor to deliver the pharmaceutical formulations ofthe present invention is also contemplated herein. As used herein, theterm “microinfusor” means a subcutaneous delivery device designed toslowly administer large volumes (e.g., up to about 2.5 mL or more) of atherapeutic formulation over a prolonged period of time (e.g., about 10,15, 20, 25, 30 or more minutes). See, e.g., U.S. Pat. Nos. 6,629,949;6,659,982; and Meehan et al., J. Controlled Release 46:107-116 (1996).Microinfusors are particularly useful for the delivery of large doses oftherapeutic proteins contained within high concentration (e.g., about100, 125, 150, 175, 200 or more mg/mL) or viscous solutions.

In one embodiment, the pharmaceutical formulation is administered via anIV drip, such that the formulation is diluted in an IV bag containing aphysiologically acceptable solution. In one embodiment, thepharmaceutical composition is a compounded sterile preparation in anintravenous infusion bag, such that a single dose of drug product isdiluted into 100 mL, 250 mL (or other like amount suitable forintravenous drip delivery) of a physiological buffer (e.g., 0.9%saline). In some embodiments, the infusion bag is made of a polyvinylchloride (e.g., VIAFLEX, Baxter, Deerfield, Ill.). In some embodiments,the infusion bag is made of a polyolefin (EXCEL IV Bags, Braun MedicalInc., Bethlehem, Pa.).

Therapeutic Uses of the Pharmaceutical Formulations

The pharmaceutical formulations of the present invention are useful,inter alia, for the treatment, prevention or amelioration of any diseaseor disorder associated with Activin A activity, including diseases ordisorders mediated by Activin A. Exemplary, non-limiting diseases anddisorders that can be treated or prevented by the administration of thepharmaceutical formulations of the present invention includeFibrodysplasia ossificans progressiva (FOP). FOP is a rare heritabledisorder in which heterotopic ossification forms histologically andbiomechanically ‘normal’ bone at extraskeletal sites, such as connectivetissue. This disorder, although episodic, is cumulative, and results inpermanent disability of increasing severity. FOP is a relentless,progressive, ultra-rare genetic disorder in which muscles, tendons andligaments are progressively replaced by bone, a process knownHeterotopic Ossification (HO). HO of the jaw, spine and rib cage canmake it difficult to speak, eat or breathe, leading to weight loss andescalating loss of mobility and skeletal deformity. People with FOP alsoexperience episodic, localized inflammation known as a “flare-ups”though HO may occur both silently as well as in association withsymptoms. Most people with FOP are wheelchair bound by 30 years old andthe median age of survival is approximately 40 years. Death oftenresults from complications, such as pneumonia, heart failure andaspiration, stemming from HO and loss of mobility in the chest, neck andjaw.

EXAMPLES

The following examples are presented so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by mole, molecular weight is averagemolecular weight, percent concentration (%) means the mass of the solutein grams divided by the volume of the solution in milliliters times 100%(e.g., 10% sucrose means 0.1 gram of sucrose per milliliter ofsolution), temperature is in degrees Centigrade, and pressure is at ornear atmospheric pressure. Initial formulation development activitiesinvolved screening organic cosolvents, thermal stabilizers, and buffersin liquid and lyophilized formulations of anti-Activin A antibodies toidentify excipients that are compatible with the protein and enhance itsstability, while maintaining near physiologic osmolality and lowviscosity for intravenous and subcutaneous injection. Buffer conditionswere also examined to determine the optimal pH for maximum proteinstability.

Example 1: Exemplary Anti-Activin A Antibody Formulation

For First in Human (FIH) studies, a lyophilized drug product (DP) wasdeveloped intended to be used for both intravenous (IV) and subcutaneous(SC) administration. The formulation for FIH was developed by assessingthe effects of different components of the formulation on the stabilityof an anti-Activin A antibody, for example, garetosmab. These componentsincluded pH, buffer, thermal stabilizers, and surfactants. The majordegradation pathways for garetosmab are formation of high and lowmolecular weight species, and acidic charge variants. Analytical methodswere developed to monitor these degradation products. In addition,general quality attributes were monitored. These included proteinconcentration, pH, solubility, and biological potency. Based on theformulation development studies executed, a lyophilized formulation wasdeveloped which included histidine at pH 6.3, sucrose and polysorbate80. The lyophilized formulation developed for FIH was stable for atleast 36 months.

Garetosmab was delivered by IV and SC administration in a Phase 1clinical study. A single, dual-use lyophilized formulation was developedsuch that lyophilized garetosmab DP was reconstituted with sterile waterfor injection (WFI) to a concentration of either 50 mg/mL garetosmab forIV infusion, or 150 mg/mL garetosmab for SC injection. Formulationdevelopment activities involved assessment of buffers, pH, organicco-solvents, surfactants, and sucrose (as the thermal stabilizer) toidentify excipients that optimize protein stability. Garetosmab DP wasproduced by lyophilization of an optimized, aqueous buffered formulationcontaining 10 mM histidine, pH 6.3, 0.1% (w/v) polysorbate 80, 5% (w/v)sucrose, and 50 mg/mL garetosmab. Table lists the composition ofgaretosmab DP after reconstitution with different volumes of WFI for IVor SC administration.

TABLE 2 Composition of Garetosmab DP for First in Human Studies AfterReconstitution with Different Volumes of WFI for IV or SC AdministrationFormulation Reconstituted for IV Reconstituted for SC ComponentAdministration Administration Garetosmab 50 mg/mL 150 mg/mL Histidine 10mM 30 mM Sucrose   5%  15% Polysorbate 80 0.1% 0.3% pH 6.3 6.3 DP, drugproduct; IV, intravenous; SC, subcutaneous; WFI, Water for Injection

The effect of buffer and pH on the thermal stability of garetosmab wasinitially examined in liquid formulations by incubating 5 mg/mLgaretosmab at 45° C. for 28 days in a series of buffer systems atvarying pH ranges. Maximum protein stability was observed whengaretosmab was formulated between pH 5.5 and 6.5 in 10 mM histidinebuffer (Table). The effect of pH on the thermal stability of garetosmabwas further examined in liquid formulations in histidine buffers rangingin pH from 6.0 to 6.6. A pH of 6.3 was selected for the DP formulationbecause the formation of high molecular weight (HMW) species was minimalat this pH (Table). This target pH would also permit a suitable pH rangefor maximal stability that could be achieved in a large-scalemanufacturing setting. Based on these results, 10 mM histidine buffer atpH 6.3 was selected for the garetosmab DP formulation.

The effect of surfactants and organic co-solvents on the agitationstress stability, freeze/thaw stability, and thermal stability of 10mg/mL garetosmab was examined in liquid formulations. The results ofthese studies are summarized in Table through Table. Garetosmab wasunstable when agitated in the absence of an organic co-solvent orsurfactant. All the organic co-solvents and surfactants that were testedprotected garetosmab from agitation-induced instability (Table). 10mg/mL garetosmab was unstable when exposed to multiple freeze/thawcycles in the absence of an organic co-solvent or surfactant (Table).All the organic co-solvents and surfactants that were tested protectedgaretosmab from freeze/thaw-induced instability (Table). In a subsequentstudy, which exposed 70 mg/mL garetosmab to eight freezing and thawingcycles, no visual failures or increases in HMW species were observed,even in solutions which did not contain surfactant or co-solvent(Table). 70 mg/mL is representative of the concentrations of garetosmabthat were subjected to freezing and thawing in a manufacturing settingduring clinical trials of garetosmab. Polysorbate 80 was selected as thesurfactant for the initial garetosmab DP formulation because itstabilized the protein to agitation stress and freeze/thaw-inducedinstability and had a minimal adverse impact on the thermal stability,while demonstrating a safe history of use in monoclonal antibodyformulations (Table through Table).

The stabilizing effect of sucrose on garetosmab was assessed. 10 mg/mLgaretosmab in a liquid formulation exhibited improved stability whenformulated with 5% sucrose, compared to no sucrose present, andincubated under accelerated conditions (Table). Garetosmab wassufficiently stable for initial clinical use when formulated in thepresence of histidine, polysorbate 80, and sucrose, at pH 6.3.

Based on the initial formulation development studies, the followingformulation was selected for clinical use: 50 mg/mL garetosmab, 10 mMhistidine, pH 6.3, 5% (w/v) sucrose, and 0.1% (w/v) polysorbate 80. TheDP was produced by filling 5.74 mL of this formulation into 20 mL glassvials and lyophilized. Research stability studies indicated that thisformulation was stable for 36 months (Table 9).

TABLE 3 Effect of Buffer and pH on the Stability of 5 mg/mL GaretosmabIncubated at 45° C. for 28 Days Formulation 5 mg/mL garetosmab, 10 mMbuffer Fill Volume 0.4 mL Container/Closure 2 mL Type 1 borosilicateglass vial with a FluroTec ®-coated 4432/50 butyl rubber stopper % TotalTurbidity Protein Change in Charge (Increase Recovered Change in Purityby Variants by in by SE-UPLC^(a) CEX-UPLC^(a) FDG Lot Color and OD atRP- % % % % % % pH/Buffer Number Appearance 405 nm) UPLC HMW Native LMWAcidic Main Basic pH 4.5, Acetate L13-322 Pass 0.00 102 2.5 −4.3 1.9−5.4 −16.4 21.8 pH 5.0, Acetate L13-323 Pass 0.00 96 1.6 −3.2 1.6 −0.9−14.6 15.5 pH 5.5, Acetate L13-324 Pass 0.00 106 1.4 −1.9 0.5 11.0 −11.30.4 pH 5.5, Histidine L13-325 Pass 0.00 92 1.2 −2.2 1.0 3.6 −7.9 4.3 pH6.0, Histidine L13-326 Pass 0.01 100 0.8 −1.8 1.0 8.7 −11.6 2.9 pH 6.5,Histidine L13-327 Pass 0.00 97 1.0 −1.3 0.3 17.2 −16.7 −0.5 pH 6.5,Phosphate L13-328 Pass 0.00 95 2.5 −2.9 0.3 20.3 −17.2 −3.0 pH 7.0,Phosphate L13-329 Pass 0.00 94 2.9 −3.6 0.7 36.2 −31.4 −4.8 ^(a)Reportedas a change in purity relative to the starting material; The startingmaterial (no incubation) contains ≥98.5% native peak by SE-UPLC and≥55.8% main peak by CEX-UPLC in all formulations. CEX, cation exchange;DS, drug substance; FDG, Formulation Development group; HMW, highmolecular weight; LMW, low molecular weight; OD, optical density; RP,reversed-phase; SE, size exclusion; UPLC, ultra performance liquidchromatography

TABLE 4 Effect of Buffer and pH on the Stability of 25 mg/mL GaretosmabIncubated at 45° C. for 28 Days Formulation 25 mg/mL garetosmab, 10 mMbuffer Fill Volume 0.5 mL Container/Closure 2 mL Type 1 borosilicateglass vial with a FluroTec ®-coated 4432/50 butyl rubber stopper % TotalTurbidity Protein Change in Charge (Increase Recovered Change in Purityby Variants by in by SE-UPLC^(a) CEX-UPLC^(a) FDG Lot Color and OD atRP- % % % % % % pH/Buffer Number Appearance 405 nm) UPLC HMW Native LMWAcidic Main Basic pH 6.0, Histidine L13-1003 Pass 0.01 95 2.3 −6.2 3.73.8 −11.0 7.2 pH 6.1, Histidine L13-1004 Pass 0.00 99 1.5 −3.6 2.0 6.2−12.8 6.6 pH 6.2, Histidine L13-1005 Pass 0.00 97 1.2 −2.7 1.4 8.8 −14.15.3 pH 6.3, Histidine L13-1006 Pass 0.00 100 1.4 −2.4 1.0 10.6 −14.6 4.0pH 6.4, Histidine L13-1007 Pass 0.00 100 1.4 −2.1 0.7 12.1 −14.7 2.6 pH6.5, Histidine L13-1008 Pass 0.01 100 1.5 −2.2 0.6 13.2 −15.6 2.5 pH6.6, Histidine L13-1009 Pass 0.01 99 1.8 −2.0 0.2 15.2 −18.1 2.9^(a)Reported as a change in purity relative to the starting material;The starting material (no incubation) contains ≥98.6% native peak bySE-UPLC and ≥64.7% main peak by CEX-UPLC in all formulations. CEX,cation exchange; DS, drug substance; FDG, Formulation Development group;HMW, high molecular weight; LMW, low molecular weight; OD, opticaldensity; RP, reversed-phase; SE, size exclusion; UPLC, ultra performanceliquid chromatography

TABLE 5 Effect of Organic Co-solvents and Surfactants on the Stabilityof 10 mg/mL Garetosmab After Agitation (120 Minutes of Vortexing)Formulation 10 mg/mL garetosmab, 10 mM histidine, pH 6.0 Fill Volume 0.4mL Container/Closure 2 mL Type 1 borosilicate glass vial with aFluorTec ®-coated 4432/50 butyl rubber stopper Turbidity % Total Changein Purity by Change in Charge Variants (Increase in Protein SE-UPL C^(a)by CEX-UPLC^(a) Co-Solvent/ FDG Lot Color and OD at Recovered by % % % %% % Surfactant Number Appearance 405 nm) pH RP-UPLC HMW Native LMWAcidic Main Basic No Co-Solvent/ L13-468 Pass 0.13 6.1 101 0.5 −0.5 −0.1−0.4 0.3 0.1 Surfactant 5% (w/v) L13-469 Pass 0.16 6.1 98 0.8 −0.8 0.0−0.4 0.3 0.1 Sucrose 0.1% (w/v) L13-470 Pass 0.00 6.1 100 −0.1 0.1 0.0−0.1 0.2 −0.1 Polysorbate 20, 5% Sucrose 0.1% (w/v) L13-471 Pass 0.006.1 100 0.0 0.0 0.0 −0.2 0.2 0.0 Polysorbate 80, 5% Sucrose 0.5% (w/v)L13-472 Pass 0.00 6.1 100 0.0 0.1 0.0 −0.2 0.8 −0.6 PEG3350, 5% Sucrose1.5% (w/v) L13-473 Pass 0.00 6.1 100 0.0 0.1 0.0 −0.2 0.3 −0.1 PEG3350,5% Sucrose ^(a)Reported as a change in purity relative to the startingmaterial; The starting material (no incubation) contains ≥98.8% nativepeak by SE-UPLC and ≥55.5% main peak by CEX-UPLC in all fiveformulations. CEX, cation exchange; DS, drug substance; FDG, FormulationDevelopment group; HMW, high molecular weight; LMW, low molecularweight; OD, optical density; RP, reversed-phase; SE, size exclusion;UPLC, ultra performance liquid chromatography

TABLE 6 Effect of Organic Co-Solvents and Surfactants on the Stabilityof 10 mg/mL Garetosmab After Eight Freezing and Thawing CyclesFormulation 10 mg/mL garetosmab, 10 mM histidine, pH 6.0 Fill Volume 0.4mL Container/Closure 2 mL Type 1 borosilicate glass vial with aFluorTec ®-coated 4432/50 butyl rubber stopper Turbidity % Total Changein Purity by Change in Charge Variants (Increase in Protein SE-UPL C^(a)by CEX-UPLC^(a) Co-Solvent/ FDG Lot Color and OD at Recovered by % % % %% % Surfactant Number Appearance 405 nm) pH RP-UPLC HMW Native LMWAcidic Main Basic No Co-Solvent/ L13-468 Fail 0.47 6.1 107 0.0^(b)0.0^(b) 0.0^(b) −0.1 0.1 0.0 Surfactant 5% (w/v) L13-469 Fail 0.40 6.197 1.0 −1.0 0.0 −0.1 0.1 0.0 Sucrose 0.1% (w/v) L13-470 Pass 0.00 6.1102 −0.1 0.1 0.0 0.0 0.1 −0.1 Polysorbate 20, 5% Sucrose 0.1% (w/v)L13-471 Pass 0.00 6.1 102 0.0 0.0 0.0 0.0 0.5 −0.5 Polysorbate 80, 5%Sucrose 0.5% (w/v) L13-472 Pass 0.00 6.1 105 −0.1 0.2 −0.1 −0.1 0.3 −0.1PEG3350, 5% Sucrose 1.5% (w/v) L13-473 Pass 0.00 6.1 104 −0.1 0.2 −0.10.2 −0.4 0.2 PEG3350, 5% Sucrose ^(a)Reported as a change in purityrelative to the starting material; The starting material (no incubation)contains ≥98.8% native peak by SE-UPLC and ≥55.5% main peak by CEX-UPLCin all five formulations. ^(b)Values for four freeze/thaw cycles are:□HMW = 0.5%, □Native = −0.4%, and □LMW = 0.0% CEX, cation exchange; DS,drug substance; FDG, Formulation Development group; HMW, high molecularweight; LMW, low molecular weight; OD, optical density; RP,reversed-phase; SE, size exclusion; UPLC, ultra performance liquidchromatography

TABLE 7 Effect of Organic Co-Solvents and Surfactants on the Stabilityof 70 mg/mL Garetosmab after Eight Freezing and Thawing CyclesFormulation 70 mg/mL garetosmab, 10 mM histidine, pH 6.0 Fill Volume 0.4mL Container/Closure 2 mL Type 1 borosilicate glass vial with aFluorTec ®-coated 4432/50 butyl rubber stopper % Total Turbidity ProteinChange in Charge (Increase Recovered Change in Purity by Variants by inby SE-UPLC^(a) CEX-UPLC^(a) Co-Solvent/ FDG Lot Color and OD at RP- % %% % % % Surfactant Number Appearance 405 nm) pH UPLC HMW Native LMWAcidic Main Basic No Co-Solvent/ L13-595 Pass 0.03 6.1 102 0.0 −0.1 0.1−0.2 0.1 0.1 Surfactant 5% (w/v) L13-596 Pass 0.02 6.1 100 −0.1 −0.1 0.2−0.3 0.3 0.0 Sucrose 0.1% (w/v) L13-597 Pass 0.01 6.1 102 −0.1 0.0 0.1−0.3 0.3 0.0 Polysorbate 80, 5% Sucrose 0.5% (w/v) L13-598 Pass 0.01 6.199 −0.1 0.0 0.1 −0.4 0.4 −0.1 PEG3350, 5% Sucrose ^(a)Reported as achange in purity relative to the starting material; The startingmaterial (no incubation) contains ≥98.6% native peak by SE-UPLC and≥56.3% main peak by CEX-UPLC in all five formulations. CEX, cationexchange; DS, drug substance; FDG, Formulation Development group; HMW,high molecular weight; LMW, low molecular weight; OD, optical density;RP, reversed-phase; SE, size exclusion; UPLC, ultra performance liquidchromatography

TABLE 8 Effect of Organic Co-Solvents and Surfactants on the Stabilityof 10 mg/mL Garetosmab Incubated at 45° C. for 28 Days Formulation 10mg/mL garetosmab, 10 mM histidine, pH 6.0 Fill Volume 0.4 mLContainer/Closure 2 mL Type 1 borosilicate glass vial with aFluorTec ®-coated 4432/50 butyl rubber stopper % Total Turbidity ProteinChange in Charge (Increase Recovered Change in Purity by Variants by inby SE-UPLC^(a) CEX-UPLC^(a) Co-Solvent/ FDG Lot Color and OD at RP- % %% % % % Surfactant Number Appearance 405 nm) pH UPLC HMW Native LMWAcidic Main Basic No Co-Solvent/ L13-468 Pass 0.00 6.2 98 0.9 −3.2 2.49.1 −12.5 3.5 Surfactant 5% (w/v) L13-469 Pass 0.00 6.1 101 0.6 −2.7 2.010.2 −13.0 2.8 Sucrose 0.1% (w/v) L13-470 Pass 0.01 6.0 101 6.1 −8.1 2.112.1 −22.2 10.1 Polysorbate 20, 5% Sucrose 0.1% (w/v) L13-471 Pass 0.016.1 101 4.2 −6.6 2.3 13.2 −24.5 11.3 Polysorbate 80, 5% Sucrose 0.5%(w/v) L13-472 Pass 0.01 6.1 101 1.2 −3.1 2.0 14.1 −17.4 3.3 PEG3350, 5%sucrose 1.5% (w/v) L13-473 Pass 0.02 6.0 103 5.7 −8.1 2.4 14.9 −28.413.5 PEG3350, 5% Sucrose ^(a)Reported as a change in purity relative tothe starting material; The starting material (no incubation) contains≥98.8% native peak by SE-UPLC and ≥55.5% main peak by CEX-UPLC in allfive formulations. CEX, cation exchange; DS, drug substance; FDG,Formulation Development group; HMW, high molecular weight; LMW, lowmolecular weight; OD, optical density; RP, reversed-phase; SE, sizeexclusion; UPLC, ultra performance liquid chromatography

TABLE 9 Development Stability of Garetosmab Lyophilized C1P2 DrugProduct Stored at 5° C. Pre-lyophilized Formulation 50 mg/mL garetosmab,10 mM histidine, pH 6.3, 5% (w/v) sucrose, 0.1% (w/v) polysorbate 80Fill Volume 5.3 mL Container/Closure 20 mL Type 1 borosilicate glassvials with a West V10-F597W 4432/50 GRY B2-TR stopper Length of Storageat 5° C. (months) Assay 0 1 3 6 9 12 18 24 36 Analysis of LyophilizedDrug Product Cake Appearance Pass Pass Pass Pass Pass Pass Pass PassPass % Moisture 0.13 NR NR 0.18 NR 0.19 NR 0.25 0.29 Reconstitution Time(seconds) 60 60 60 90 70 60 60 60 120 Analysis of Reconstituted DrugProduct^(a) Color and Appearance Pass Pass Pass Pass Pass Pass Pass PassPass Turbidity (Increase in OD at 405 nm) 0.00 0.01 0.00 0.00 0.00 0.000.00 0.00 0.00 pH 6.4 6.3 6.3 6.3 6.3 6.3 6.3 6.3 6.3 Particulate 2 to10 μm 1168 NR NR 2744 NR 1122 NR 1941 6357 Analysis by ≥10 μm 15 NR NR48 NR 17 NR 6 121 MFI ≥25 μm 7 NR NR 10 NR 4 NR 0 7 % Total ProteinRecovered by 100 94 96 93 94 95 95 96 94 RP-UPLC Purity by Non-reduced;100 NR NR 100 NR 100 NR 100 100 MCE-SDS % main peak Reduced; 100 NR NR100 NR 100 NR 100 100 % heavy + light chain Purity by % HMW 0.8 0.8 0.80.9 0.9 0.9 0.9 1.0 1.2 SE-UPLC % Native 98.7 98.8 98.6 98.5 98.5 98.598.5 98.3 97.8 % LMW 0.5 0.4 0.6 0.6 0.6 0.6 0.6 0.7 1.1 Charge % Acidic31.4 31.6 30.7 31.4 31.5 31.7 32.7 32.0 32.2 Variant % Main 53.4 52.551.3 52.3 51.6 52.7 50.2 51.6 52.4 Analysis by % Basic 15.1 15.9 18.016.3 17.0 15.6 17.1 16.4 15.4 CEX-UPLC Charge % Acidic 48.4 NR NR 44.6NR 48.5 NR 49.8 49.0 Variant % Main 48.4 NR NR 51.5 NR 46.3 NR 43.6 45.7Analysis by % Basic 3.2 NR NR 3.9 NR 5.1 NR 6.6 5.3 iCIEF % RelativePotency by Bioassay 103 NR NR 114 NR 135 NR 106 95 ^(a)Samples werereconstituted with sterile Water for Injection to 100 mg/mL garetosmab.C1P2, cell line 1 process 2; CEX, cation exchange; DS, drug substance;FDG, Formulation Development group; HMW, high molecular weight; iCIEF,imaged capillary isoelectric focusing; LMW, low molecular weight;MCE-SDS, microchip capillary electrophoresis-sodium dodecyl sulfate;MFI, Micro-Flow Imaging ™; NR, not required; OD, optical density; RP,reversed-phase; SE, size exclusion; UPLC, ultra performance liquidchromatography.

Example 2:Exemplary Anti-Activin A Antibody Formulations

The objective of the drug product (DP) commercial development activitieswere to develop an anti-Activin A antibody (e.g., garetosmab) DP withthe following attributes: a liquid formulation in glass vials with aconcentration of garetosmab sufficient to deliver a dose of 10 mg/kggaretosmab by intravenous (IV) infusion; the vial of garetosmab shouldcontain 300 mg in 5 mL; a near iso-osmolar formulation that can bediluted for IV infusion with commonly available diluents; a formulationthat is compatible with and stable in type 1 clear glass vials andstandard serum stopper as the primary packaging system; a sterile DPthat supports long-term stability with a shelf-life of 24 months orlonger at 2-8° C.; a robust formulation that minimizes formation ofgaretosmab high molecular weight (HMW) species, minimizes changes in therelative distribution of garetosmab charge variant species, minimizesthe presence of visible and subvisible particles, and maintains thebiological activity when subjected to handling and thermal stresses; anda formulation that will stabilize high garetosmab concentrations andhave characteristics that are acceptable for use in subcutaneous (SC)administration.

This study describes the late phase formulation optimization, garetosmabcommercial formulation confirmation, and garetosmab robustness. Thegaretosmab commercial formulation is a liquid containing 60 mg/mLgaretosmab in an aqueous solution containing 10 mM L-histidine, pH 6.3,5% (w/v) sucrose, 70 mM L-arginine HCl, and 0.05% (w/v) polysorbate 20.This formulation stabilizes garetosmab during long-term storage at 2-8°C. and when exposed to accelerated and stress conditions, includingagitation, freezing and thawing, and thermal stress. For Phase 2clinical studies and commercialization, a liquid DP formulation suitablefor IV administration was developed. This formulation was built uponknowledge gained during initial development, plus additional formulationdevelopment studies performed to refine and optimize the formulation forthe specific clinical indication and to optimize product stability. Theformulation for garetosmab consists of the following: 60 mg/mLgaretosmab, 10 mM histidine, pH 6.3, 5% sucrose, 70 mM arginine-HC1, and0.05% polysorbate 20. The DP is manufactured by filling 5.61 mL into a10 mL glass vial and sealed with an elastomeric stopper. Long-termstability studies have been initiated and indicate that the formulationis stable for at least 12 months. The stability studies will continuefor 60 months. Robustness studies have been initiated. Long-term,accelerated and stress stability studies thus far indicate that theformulation is robust with respect to pH, protein concentration andexcipient concentration. Small variations that might occur in themanufacturing process have no meaningful impact to the quality ofgaretosmab DP.

The pharmaceutical development studies executed during the garetosmabdevelopment process resulted in a stable formulation for garetosmab thatmeets the goals set forth to address the clinical and commercial needs.The goal for late-stage clinical development was to produce aformulation that would deliver 300 mg of garetosmab in 5 mL of solution.A secondary goal was to have a formulation that would be easilyconverted to a subcutaneous formulation for future use. Formulationdevelopment studies were conducted with the goal of developing a liquidformulation containing 60 mg/mL garetosmab that will be used for IVadministration. Formulation development activities for the liquidformulation involved assessment of histidine buffer concentration,surfactant concentration, and other stabilizers.

Selection of Buffer and pH for the Garetosmab Liquid Formulation

Initial FIH development activities determined that the optimal pH forgaretosmab was 6.3. The pH remained unchanged throughout development.Histidine buffer was utilized for the lyophilized formulation forinitial Phase 1 studies of garetosmab based upon a buffer screeningstudy. The same buffer was chosen for late stage formulationdevelopment. The stability of 50 mg/mL garetosmab was tested whenincubated at 45° C. for 28 days at pH 6.3 when the histidineconcentration was 5 mM, 10 mM or 25 mM to assess the optimal histidineconcentration. The results of this stress test indicate that 10 mMhistidine at pH 6.3 falls in a stable region of the design space withsufficient space on either end to tolerate small variations in thebuffer concentration (Table) or pH (Table). Based on these results thesame buffer composition and pH used for the lyophilized formulation,histidine, at a concentration of 10 mM and a pH of 6.3, was selected asthe buffer, buffer concentration, and pH for the late stage liquidformulation.

Selection of Thermal Stabilizers for the Garetosmab Liquid Formulation

Initial development studies for garetosmab demonstrated that 5% (w/v)sucrose was required to stabilize garetosmab to freeze-thaw stress.Therefore, 5% (w/v) sucrose was added to the bulk Drug Substance (DS)because it adequately stabilizes the bulk DS against the formation ofHMW species when subjected to freezing and thawing cycles that the DSwould be exposed to during manufacturing (freezing and thawing stabilityfor DS is discussed in Module S.7.1). A study was performed to assessthe stability of 50 mg/mL liquid garetosmab when incubated at 45° C. for28 days or subjected to 8 cycles of freezing and thawing, when thesucrose concentration in the formulation was varied between 0 and 10%.The results of these stress tests indicate that 5% sucrose falls in astable region of the design space with sufficient space on either end totolerate small variations in the sucrose concentration that may beexperienced as a result of normal manufacturing variability (Table andTable). 5% sucrose was chosen as a stabilizer because it sufficientlystabilizes garetosmab to thermal, and freezing and thawing stresses andprovides an acceptable osmolality in the final formulation.

Arginine was evaluated as an additional stabilizer for the garetosmabliquid formulation. During the assessment, a range of arginineconcentrations from 0 to 100 mM was evaluated (Table). The results ofthe stress test indicates that 70 mM arginine falls in a stable regionof the design space with sufficient space on either end to toleratesmall variations in the arginine concentration that may be experiencedas a result of normal manufacturing variability (Table). 70 mM argininewas chosen as the stabilizer concentration in the garetosmab liquidformulation due to the reduced formation of HMW species and acidiccharge variants observed under stress stability conditions and becauseit provides an acceptable osmolality in the final formulation.

Selection of a Surfactant for the Garetosmab Liquid Formulation

0.1% (w/v) polysorbate 80 was chosen for the garetosmab Phase 1formulation. Further testing, comparing the stability of 50 mg/mLgaretosmab in the presence of polysorbate 20 or polysorbate 80,indicated that both surfactants provided comparable stability to 50mg/mL garetosmab when incubated at 45° C. for 28 days or at 25° C. for 6months (Table). Polysorbate 20 was chosen for further development of agaretosmab liquid formulation. The stability of 50 mg/mL garetosmab wasassessed when agitated for 30 minutes or incubated at 45° C. for 28 daysat varying polysorbate 20 concentrations. The results of the stress testindicates that 0.05% polysorbate 20 falls in a stable region of thedesign space with sufficient space on either end to tolerate smallvariations in the polysorbate 20 concentration that may be experiencedas a result of normal manufacturing variability (Table and Table). Apolysorbate 20 concentration of 0.05% (w/v) was chosen for thegaretosmab liquid formulation.

Conclusions

Results of studies testing garetosmab stability when formulations weresubjected to stress conditions, incubation at 45° C., vortex agitationand freeze/thaw cycling, were used to select the late-stage formulation.Based on data collected from these studies a garetosmab formulationcontaining 10 mM histidine, pH 6.3, 5% sucrose, 70 mM arginine-HCl and0.05% polysorbate 20 was selected and subsequently demonstrated to besuitable for clinical or commercial use. The excipient concentrationswere chosen such that small variations that may be experienced duringnormal manufacturing will have no observable impact on the stability orquality of the garetosmab DP. The selected formulation contains 60 mg/mLgaretosmab to provide a 300 mg dose in a withdrawable volume of 5 mL.The suitability of this formulation has been confirmed based on 18months of long-term development stability data at 5° C., 6 months ofaccelerated stability data at 25° C./60% RH and stress stability testingincluding 56 days of stability data at 40° C./75% RH, 8 cycles offreezing and thawing and 120 minutes of agitation (vortex).

TABLE 10 Effect of Histidine Concentration on the Stability of 50 mg/mLGaretosmab Incubated at 45° C. for 28 Days Formulation 50 mg/mLgaretosmab, pH 6.3 Fill Volume 0.3 mL Container/Closure 2 mL Type 1borosilicate glass vial with a FluroTec ® coated 4432/50 butyl rubberstopper % Total Change in Purity Change in Charged Variants TurbidityProtein by SE-UPLC^(a) by CEX-UPLC^(a) Histidine Lot Color and (Increasein OD Recovered by % % % % % % concentration Number Appearance at 405nm) pH RP-UPLC HMW Native LMW Acidic Main Basic  5 mM L16-1298 Pass 0.016.4 104 3.2 −3.7 0.5 13.5 −15.2 1.7 10 mM L16-1299 Pass 0.01 6.4 103 2.9−3.5 0.6 13.5 −14.6 1.2 25 mM L16-1300 Pass 0.01 6.4 103 2.6 −3.2 0.613.5 −14.6 1.1 ^(a)Reported as a change in purity relative to thestarting material. The starting material (no incubation) contains ≥98.6%native peak by SE-UPLC and ≥50.3% main peak by CEX-UPLC in allformulations. Negative numbers indicate a decrease in the measuredattribute and positive numbers indicate an increase.

TABLE 11 Effect of Sucrose Concentration on the Stability of 50 mg/mLGaretosmab Incubated at 45° C. for 28 Days Formulation 50 mg/mLgaretosmab, 10 mM histidine, pH 6.3 Fill Volume 0.3 mL Container/Closure2 mL Type 1 borosilicate glass vial with a FluroTec ® coated 4432/50butyl rubber stopper % Total Change in Purity Change in Charged VariantsTurbidity Protein by SE-UPLC^(a) by CEX-UPLC ^(a) Sucrose Lot Color and(Increase in OD Recovered % % % % % % concentration Number Appearance at405 nm) pH by RP-UPLC HMW Native LMW Acidic Main Basic   0% L16-1306Pass 0.00 6.4 100 3.0 −3.5 0.5 13.2 −14.0 0.8 2.5% L16-1307 Pass 0.006.4 102 2.7 −3.2 0.6 13.0 −13.4 0.4   5% L16-1308 Pass 0.00 6.3 101 2.5−3.0 0.5 13.2 −13.9 0.7 7.5% L16-1309 Pass 0.00 6.3 101 2.5 −3.0 0.513.9 −14.5 0.6  10% L16-1310 Pass 0.00 6.3 102 2.2 −2.8 0.6 13.2 −14.71.5 ^(a)Reported as a change in purity relative to the startingmaterial. The starting material (no incubation) contains ≥98.8% nativepeak by SE-UPLC and ≥50.2% main peak by CEX-UPLC in all formulations.Negative numbers indicate a decrease in the measured attribute andpositive numbers indicate an increase.

TABLE 12 Effect of Sucrose Concentration on the Stability of 50 mg/mLGaretosmab After 8 Cycles of Freezing and Thawing Formulation 50 mg/mLgaretosmab, 10 mM histidine, pH 6.3 Fill Volume 0.3 mL Container/Closure2 mL Type 1 borosilicate glass vial with a FluroTec ® coated 4432/50butyl rubber stopper Turbidity % Total Change in Purity Change inCharged Variants (Increase Protein by SE-UPLC^(a) by CEX-UPLC^(a)Sucrose Lot Color and in OD Recovered by % % % % % % concentrationNumber Appearance at 405 nm) pH RP-UPLC HMW Native LMW Acidic Main Basic  0% L16-1306 Pass 0.00 6.3 98 0.7 −0.9 0.2 −0.7 0.7 0.0 2.5% L16-1307Pass 0.00 6.3 98 0.1 −0.3 0.2 −0.5 0.5 0.0   5% L16-1308 Pass 0.00 6.398 0.1 −0.2 0.2 −0.6 0.0 0.5 7.5% L16-1309 Pass 0.00 6.3 98 0.0 −0.2 0.2−0.6 0.5 0.1  10% L16-1310 Pass 0.00 6.3 98 0.1 −0.2 0.2 −0.6 0.1 0.5^(a)Reported as a change in purity relative to the starting material.The starting material (no incubation) contains ≥98.8% native peak bySE-UPLC and ≥50.2% main peak by CEX-UPLC in all formulations. Negativenumbers indicate a decrease in the measured attribute and positivenumbers indicate an increase.

TABLE 13 Effect of Arginine Concentration on the Stability of 50 mg/mLGaretosmab Incubated at 45° C. for 28 Days Formulation 50 mg/mLgaretosmab, 10 mM histidine, pH 6.3 Fill Volume 0.3 mL Container/Closure2 mL Type 1 borosilicate glass vial with a FluroTec ® coated 4432/50butyl rubber stopper % Total Change Change in Turbidity Protein inPurity Charged Variants (Increase Recovered by SE-UPLC^(a) by CEX-UPLC^(a) Arginine Lot Color and in OD by % % % % % % concentration NumberAppearance at 405 nm) pH RP-UPLC HMW Native LMW Acidic Main Basic   0 mML16-1403 Pass 0.00 6.4 102 3.0 −3.6 0.6 13.2 −14.4 1.2  25 mM L16-1311Pass 0.00 6.4 102 2.3 −2.9 0.6 11.1 −12.5 1.5  50 mM L16-1312 Pass 0.006.4 100 2.3 −2.9 0.6 10.6 −13.7 3.1  70 mM L16-1313 Pass 0.00 6.3 99 2.2−2.8 0.6 10.3 −12.9 2.6 100 mM L16-1314 Pass 0.00 6.4 98 2.3 −3.0 0.79.7 −11.5 1.8 ^(a)Reported as a change in purity relative to thestarting material. The starting material (no incubation) contains ≥98.8%native peak by SE-UPLC and ≥50.3% main peak by CEX-UPLC in allformulations. Negative numbers indicate a decrease in the measuredattribute and positive numbers indicate an increase.

TABLE 14 Stability of 50 mg/mL Garetosmab Liquid Formulation in thePresence of 0.05% Polysorbate 20 or 0.05% Polysorbate 80 Incubated at45° C. for 28 Days or 25° C. for 6 Months Formulation 50 mg/mLgaretosmab, 10 mM Histidine, pH 6.3, 5% sucrose, 70 mM arginine FillVolume 0.5 mL Container/Closure 2 mL Type 1 borosilicate glass vial witha FluroTec ® coated 4432/50 butyl rubber stopper Turbidity % TotalChange in Purity Change in Charged (Increase Protein by SE-UPLC^(a)Variants by CEX-UPLC^(a) Lot Color and in OD Recovered by % % % % % %Formulation Number Appearance at 405 nm) pH RP-UPLC HMW Native LMWAcidic Main Basic 45° C. 0.05% L13-883 Pass 0.01 6.4 104 2.2 −2.9 0.67.0 −10.6 3.6 Polysorbate 20, 28 days 45° C. 0.05% L13-884 Pass 0.01 6.4104 2.1 −2.7 0.6 9.5 −13.6 4.1 Polysorbate 80, 28 days 25° C. 0.05%L13-883 Pass 0.01 6.4 102 0.1 −0.4 0.3 2.8 −8.2 5.4 Polysorbate 20, 6months 25° C. 0.05% L13-884 Pass 0.01 6.4 103 0.2 −0.1 −0.1 3.7 −10.26.5 Polysorbate 80, 6 months ^(a)Reported as a change in purity relativeto the starting material. The starting material (no incubation) contains≥98.9% native peak by SE-UPLC and ≥55.5% main peak by CEX-UPLC in allformulations. Negative numbers indicate a decrease in the measuredattribute and positive numbers indicate an increase.

TABLE 15 Effect of Polysorbate 20 Concentration on the Stability of 50mg/mL Garetosmab After Agitation for 30 Minutes Formulation 50 mg/mLgaretosmab, 10 mM histidine, pH 6.3, x % polysorbate 20 Fill Volume 0.5mL Container/Closure 2 mL Type 1 borosilicate glass vial with aFluroTec ® coated 4432/50 butyl rubber stopper % Total Change in PurityChange in Charged Variants Turbidity Protein by SE-UPLC^(a) byCEX-UPLC^(a) Polysorbate 20 Lot Color and (Increase in OD Recovered by %% % % % % concentration Number Appearance at 405 nm) pH RP-UPLC HMWNative LMW Acidic Main Basic 0.025% L16-1302 Pass 0.00 6.3 100 0.0 0.00.0 0.0 0.4 −0.5  0.05% L16-1303 Pass 0.00 6.3 100 0.0 −0.1 0.0 0.1 0.2−0.3 0.075% L16-1304 Pass 0.00 6.3 100 0.0 0.0 0.0 −0.1 −0.2 0.3  0.1%L16-1305 Pass 0.00 6.4 100 0.0 −0.1 0.1 0.0 0.0 0.0 ^(a)Reported as achange in purity relative to the starting material. The startingmaterial (no incubation) contains ≥98.8% native peak by SE-UPLC and≥50.1% main peak by CEX-UPLC in all formulations. Negative numbersindicate a decrease in the measured attribute and positive numbersindicate an increase.

TABLE 16 Effect of Polysorbate 20 Concentration on the Stability of 50mg/mL Garetosmab Incubated at 45° C. for 28 Days Formulation 50 mg/mLgaretosmab, 10 mM histidine, pH 6.3, x % polysorbate 20 Fill Volume 0.5mL Container/Closure 2 mL Type 1 borosilicate glass vial with aFluroTec ® coated 4432/50 butyl rubber stopper % Total Change in PurityChange in Charged Variants Turbidity Protein by SE-UPLC^(a) byCEX-UPLC^(a) Polysorbate 20 Lot Color and (Increase in OD Recovered by %% % % % % concentration Number Appearance at 405 nm) pH RP-UPLC HMWNative LMW Acidic Main Basic 0.025% L16-1302 Pass 0.01 6.4 103 3.2 −3.80.6 13.7 −14.7 1.0  0.05% L16-1303 Pass 0.01 6.4 104 3.1 −3.7 0.6 13.5−13.9 0.4 0.075% L16-1304 Pass 0.01 6.4 103 3.1 −3.6 0.6 13.2 −13.7 0.4 0.1% L16-1305 Pass 0.01 6.4 103 3.1 −3.7 0.6 13.6 −15.8 2.2^(a)Reported as a change in purity relative to the starting material.The starting material (no incubation) contains ≥98.8% native peak bySE-UPLC and ≥50.1% main peak by CEX-UPLC in all formulations. Negativenumbers indicate a decrease in the measured attribute and positivenumbers indicate an increase.

Example 3: Stability Studies

The suitability of the garetosmab liquid formulation developed andintended for late-stage clinical and commercial use was confirmed withlong-term, accelerated and stress stability studies. In addition, therobustness of the commercial formulation was confirmed using aDesign-Of-Experiment (DOE) approach.

Studies were initiated to evaluate the storage, accelerated and stressstability of a research lot of garetosmab DP. The FDS lot used for thesestudies is representative of the FDS manufactured for clinical use. TheDP configuration used in these studies was identical to the DPconfiguration used in Phase 2 clinical studies and was produced byfilling 5.5 mL of FDS into 10 mL Type 1 glass vials.

Research Storage, Accelerated and Stress Stability for Garetosmab DrugProduct

Garetosmab DP was physically and chemically stable when stored at 2-8°C. for at least 12 months, 18 months or 24 months (

Table). No appreciable changes were detected in any of the monitoredattributes. Results from the analysis of the garetosmab DP afterincubation under stress and accelerated temperature conditions areprovided in Table. After incubation for 56 days at 40° C./75% RH, someformation of HMW species, LMW species and charge variants (increasedrelative percentage of acidic species (Region 1)) were detected.Following incubation at 25° C./60% RH for 6 months, no increase in HMWspecies was observed by SE-UPLC, but some increases in acidic species(Region 1) were observed by CEX-UPLC and icIEF. Furthermore, smallincreases in LMW species were observed by non-reduced and reduced MCE,respectively, after incubation for 6 months at 25° C./60% RH. Nomeaningful changes in any of the other monitored attributes wereobserved. The liquid DP will be exposed to room temperature conditionsfor only short periods of time. Temperatures above room temperature willbe avoided.

Results from the analysis of the garetosmab DP after being subjected toagitation or multiple cycles of freezing and thawing are shown in Table.No meaningful changes in any of the monitored attributes were observedwhen garetosmab DP was subjected to 120 minutes of agitation or 8 cyclesof freezing and thawing. The results of this research stability studyconfirmed that the garetosmab formulation developed for commercial useis suitable and provides a stable drug product.

TABLE 17 Research Stability of Garetosmab Liquid Drug Product Stored at2-8° C. Formulation 60 mg/mL garetosmab, 10 mM histidine, pH 6.3, 70 mMarginine-HCl, 5% sucrose, 0.05% polysorbate 20 Fill Volume 5.5 mLContainer/Closure 10 mL Type 1 borosilicate glass vials with a WestS10-F451W 4432/50 GRY B2-40 stopper Length of Storage at 2-8° C.(months) Assay 0 1 2 3 6 9 12 18 24 Color and Appearance Pass Pass PassPass Pass Pass Pass Pass Pass Turbidity (Increase in OD 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 at 405 nm) pH 6.3 6.3 6.3 6.4 6.3 6.3 6.46.3 6.4 Particulate ≥10 μm 33 NR NR NR 11 NR 61 28 28 Analysis by ≥25 μm0 NR NR NR 0 NR 0 0 0 HIAC (particles per container) Particulate 2 to 10μm 59 NR NR NR 940 NR 2303 1664 2934 Analysis by ≥10 μm 5 NR NR NR 12 NR9 4 3 MFI ≥25 μm 1 NR NR NR 3 NR 3 2 2 (particles per mL) % TotalProtein Recovered by 100 101 106 101 101 96 100 98 100 RP-UPLC Non- %Main Peak 96.6 NR NR NR 96.1 NR 96.7 96.1 96.5 reduced Purity MCE % LMWSpecies 3.0 NR NR NR 3.4 NR 3.2 3.5 3.1 % HMW Species 0.5 NR NR NR 0.5NR 0.2 0.5 0.4 Reduced % Purity 94.7 NR NR NR 94.6 NR 95.0 94.2 94.5 MCE% LMW Species 1.9 NR NR NR 2.1 NR 1.8 2.5 2.0 % NGHC 1.5 NR NR NR 1.5 NR1.5 1.7 1.5 Purity by % HMW species 0.8 0.7 0.7 0.7 0.7 0.7 0.7 0.7 0.7SE-UPLC % Main peak 98.7 98.6 98.8 98.8 98.2 98.6 98.4 98.1 98.1 purity% LMW species 0.5 0.7 0.5 0.5 1.1 0.7 0.9 1.2 1.2 Charge % Region 1 24.124.2 24.1 23.7 24.4 24.6 22.4 24.4 24.6 Variant % Region 2 55.6 57.557.7 58.2 57.5 58.1 57.9 56.9 57.5 Analysis by % Region 3 20.3 18.3 18.218.2 18.1 17.4 19.7 18.7 17.9 CEX- UPLC Charge % Region 1 40.6 NR NR NR40.9 NR 40.0 40.6 42.4 Variant % Region 2 52.8 NR NR NR 52.3 NR 52.752.1 51.3 Analysis by % Region 3 6.6 NR NR NR 6.9 NR 7.3 7.3 6.3 iCIEF %Relative Potency by Bioassay 111 NR NR NR 110 NR 106 95 99 CEX, cationexchange; DS, drug substance; FDG, Formulation Development group; HMW,high molecular weight; iCIEF, imaged capillary isoelectric focusing;LMW, low molecular weight; MCE, microchip capillary electrophoresis;MFI, Micro-Flow Imaging ™; NR, not required; OD, optical density; RP,reversed-phase; SE, size exclusion; UPLC, ultra- performance liquidchromatography

TABLE 18 Research Stability of Garetosmab Liquid Drug Product StoredUnder Accelerated Conditions Formulation 60 mg/mL garetosmab, 10 mMhistidine, pH 6.3, 70 mM arginine-HC1, 5% sucrose, 0.05% polysorbate 20Fill Volume 5.5 mL Container/Closure 10 mL Type 1 borosilicate glassvials with a West S10-F451W 4432/50 GRY B2-40 stopper No 25° C./60% RHStorage Storage (months) 40° C./75% RH Storage (days) Assay T = 0 1 2 36 7 14 28 56 Color and Appearance Pass Pass Pass Pass Pass Pass PassPass Pass Turbidity (Increase in 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.010.00 OD at 405 nm) pH 6.3 6.4 6.3 6.4 6.3 6.3 6.4 6.4 6.3 Particulate≥10 33 NR NR 28 6 NR NR 6 61 Analysis by μm HIAC ≥25 0 NR NR 0 0 NR NR 00 (particles per μm container) Particulate 2 to 59 NR NR 774 1468 NR NR641 308 Analysis by 10 μm MFI (particles ≥10 5 NR NR 7 10 NR NR 19 9 permL) μm ≥25 1 NR NR 3 1 NR NR 5 1 μm % Total Protein 100 102 106 101 102101 101 101 102 Recovered by RP-UPLC Non-reduced % Main 96.6 NR NR 96.395.2 NR NR 95.6 93.7 MCE Peak Purity % 3.0 NR NR 3.3 4.3 NR NR 3.9 5.6LMW Species % 0.5 NR NR 0.4 0.5 NR NR 0.6 0.7 HMW Species Reduced % 94.7NR NR 93.4 92.3 NR NR 93.6 91.5 Purity MCE % 1.9 NR NR 2.9 4.0 NR NR 2.94.6 LMW Species % 1.5 NR NR 1.5 1.6 NR NR 1.6 1.7 NGH C Purity by % 0.80.7 0.7 0.8 0.8 0.8 0.9 1.1 1.6 SE-UPLC HMW species % 98.7 98.6 98.698.6 98.1 98.6 98.5 98.2 97.5 Main peak purity % 0.5 0.7 0.7 0.6 1.1 0.60.7 0.8 0.9 LMW species Charge % 24.1 24.4 25.2 25.8 29.6 24.3 26.1 30.639.1 Variant Region 1 Analysis by % 55.6 57.3 56.9 56.6 53.3 58.0 56.652.1 45.1 CEX-UPLC Region 2 % 20.3 18.3 17.9 17.5 17.1 17.7 17.3 17.315.7 Region 3 Charge % 40.6 NR NR 44.4 48.9 NR 44.4 51.6 60.0 VariantRegion 1 Analysis by % 52.8 NR NR 48.1 45.2 NR 48.8 42.6 34.5 iCIEFRegion 2 % 6.6 NR NR 7.6 6.0 NR 6.8 5.7 5.5 Region 3 CEX, cationexchange; DS, drug substance; FDG, Formulation Development group HMW,high molecular weight; iCIEF, imaged capillary isoelectric focusing;LMW, low molecular weight; MCE, microchip capillary electrophoresis;MFI, Micro-Flow Imaging ™; NR, not required; OD, optical density; RH,relative humidity; RP, reversed-phase; SE, size exclusion; UPLC, ultra-performance liquid chromatography

TABLE 19 Research Stability of Garetosmab Liquid Drug Product toAgitation and Freezing and Thawing Stress Formulation 60 mg/mLgaretosmab, 10 mM histidine, pH 6.3, 70 mM arginine-HCl, 5% sucrose,0.05% polysorbate 20 Fill Volume 5.5 mL Container/Closure 10 mL Type 1borosilicate glass vials with a West S10-F451W 4432/50 GRY B2-40 stopper#Freezing and Thawing No Storage Agitation by Vortex (minutes) CyclesAssay T = 0 30 60 120 4 8 Color and Appearance Pass Pass Pass Pass PassPass Turbidity (Increase 0.00 0.00 0.00 0.00 0.00 0.00 in OD at 405 nm)pH 6.3 6.3 6.3 6.3 6.3 6.3 Particulate ≥10 μm 33 NR NR 66 NR 77 Analysisby ≥25 μm 0 NR NR 11 NR 6 HIAC (particles per container) Particulate 2to 10 μm 59 NR NR 1362 NR 695 Analysis by ≥10 μm 5 NR NR 13 NR 10 MFI(particles ≥25 μm 1 NR NR 3 NR 3 per mL) % Total Protein 100 101 101 100101 103 Recovered by RP-UPLC Non- % Main Peak Purity 96.6 NR NR 96.5 NR96.7 reduced % LMW Species 3.0 NR NR 3.1 NR 2.9 MCE % HMW Species 0.5 NRNR 0.4 NR 0.5 Reduced % Purity 94.7 NR NR 94.8 NR 95.3 MCE % LMW Species1.9 NR NR 1.9 NR 1.6 % NGHC 1.5 NR NR 1.6 NR 1.4 Purity % HMW species0.8 0.8 0.7 0.8 0.8 0.8 by % Main peak purity 98.7 98.7 98.6 98.6 98.798.6 SE-UPLC % LMW species 0.5 0.6 0.6 0.6 0.6 0.6 Charge % Region 124.1 24.2 23.3 24.2 23.6 23.6 Variant % Region 2 55.6 55.7 56.4 55.558.5 58.6 Analysis by % Region 3 20.3 20.1 20.3 20.3 17.9 17.8 CEX-UPLCCharge % Region 1 40.6 NR NR 42.1 NR 40.7 Variant % Region 2 52.8 NR NR51.2 NR 51.4 Analysis by % Region 3 6.6 NR NR 6.7 NR 7.9 iCIEF CEX,cation exchange; DS, drug substance; FDG, Formulation Development group;HMW, high molecular weight; iCIEF, imaged capillary isoelectricfocusing; LMW, low molecular weight; MCE, microchip capillaryelectrophoresis; MFI, Micro-Flow Imaging ™; NR, not required; OD,optical density; RH, relative humidity; RP, reversed-phase; SE, sizeexclusion; UPLC, ultra- performance liquid chromatography

Conclusions

The results from the long-term storage, accelerated, and stressstability studies of the garetosmab DP support the clinical use of thisproduct. garetosmab DP can withstand short exposures to room temperaturewithout compromising physical or chemical stability. Garetosmab DP willbe stored at 2° C. to 8° C. and exposure to temperatures greater than 2°C. to 8° C. will be limited.

Example 4: Robustness Studies

Normal variation in the composition of garetosmab DP may occur duringmanufacturing, including variations in the concentration of garetosmab,the concentration of the excipients (histidine, arginine, sucrose, orpolysorbate 20), and/or the pH of the formulation. Because variations inany of these formulation factors could potentially impact the stabilityor quality of the DP, formulation robustness studies were conducted toassess the impact of such variations, within defined ranges.

Two DOE studies were used to evaluate the effect of each individualformulation factor as well as the influence of interactions betweenfactors on the formulation stability:

Preliminary robustness study—DOE study with accelerated and stressstability assessment to identify the key formulation factors that mayimpact garetosmab DP stability.

Final robustness study—a comprehensive DOE study evaluating the keyformulation factors identified from the preliminary robustness study,with stress, accelerated, and long-term shelf-life stability to evaluategaretosmab formulation robustness.

The nominal composition of the garetosmab commercial formulationconsists of 60 mg/mL Garetosmab, 10 mM histidine, 5% (w/v) sucrose, 70mM arginine-HCl, 0.05% (w/v) polysorbate 20, at pH 6.3.

The purpose of this preliminary robustness study was to examine maineffects and the combined effects of several factors (interactions andquadratics) on the stability of garetosmab and identify factors thatshould be included in a comprehensive robustness study (main effects arethe formulation factors, interactions describe interactions between twodifferent factors, and quadratics describe interactions where a factorinteracts with itself). All main effects were examined in this study. Asubset of interactions and quadratics were chosen to be examined basedon a risk assessment informed by prior experience with garetosmab. Basedon the risk assessment a 6-factor D-optimal DOE study was developed tocharacterize and explore the excipient design space. A 24-run study wasdeveloped which considered main effects, and the interactions andquadratics that were defined in the risk assessment. The design wascreated, evaluated and confirmed in JMP12.1 to be properly powered andable to cover the design space with good estimation capability. Thepreliminary robustness study design is described in Table, Table, andTable.

TABLE 20 Factors tested in the garetosmab preliminary robustness studyVariation Formulation factor Target level Range tested from TargetGaretosmab 60 mg/ml 54-66 mg/mL ±10% Concentration Sucrose Concentration  5% 4-6% ±20% Arginine Concentration 70 mM 56-84 mM ±20% HistidineConcentration 10 mM 8-12 mM ±20% pH 6.3 6.0-6.6 ±0.3 pH unitsPolysorbate 20 0.05% 0.025-0.075% ±50% concentration

TABLE 21 Formulations assessed in the garetosmab preliminary robustnessstudy Formulation Protein, Histidine, Sucrose, Arginine, Polysorbate #Lot # mg/mL mM pH % (w/v) mM 20, % (w/v) F1 L18-2712 66 8 6.6 6 56 0.025F2 L18-2713 54 8 6.6 6 84 0.075 F3 L18-2714 66 10 6.6 6 70 0.075 F4L18-2715 54 8 6.0 4 84 0.025 F5 L18-2716 54 10 6.6 4 84 0.025 F6L18-2717 66 8 6.0 6 84 0.075 F7 L18-2718 66 12 6.6 4 56 0.075 F8L18-2719 66 8 6.6 4 84 0.025 F9 L18-2720 66 8 6.0 4 56 0.075 F10L18-2721 54 12 6.3 4 84 0.025 F11 L18-2722 54 10 6.3 6 56 0.075 F12L18-2723 66 10 6.0 4 70 0.025 F13 L18-2724 54 8 6.6 4 56 0.075 F14L18-2725 54 12 6.6 4 70 0.075 F15 L18-2726 66 12 6.6 6 84 0.075 F16L18-2727 54 12 6.0 6 84 0.075 F17 L18-2728 54 12 6.6 6 56 0.025 F18L18-2729 54 12 6.0 4 56 0.075 F19 L18-2730 66 8 6.3 4 70 0.075 F20L18-2731 66 12 6.0 4 84 0.025 F21 L18-2732 54 8 6.0 6 56 0.025 F22L18-2733 54 8 6.0 6 70 0.025 F23 L18-2734 66 12 6.0 6 56 0.025 F24L18-2735 66 12 6.3 6 70 0.025

TABLE 22 Storage conditions and sampling intervals for garetosmabpreliminary robustness study Incubation Temperature Sampling intervals25° C. 1, 3, 6 months 45° C. 7, 14, 21, 28 days t = 0 0 (days)

Results

In the preliminary garetosmab robustness study, the stability ofgaretosmab as a function of varying formulation was assessed at both 25°C. and 45° C. However, the factors chosen for the final robustness studywere based on the rates of degradation determined at 45° C. only. If therate of change of a response had p≤0.05 for a specific factor (includingmain effects, interactions and quadratics), that factor was consideredstatistically significant and would be considered for the finalrobustness study. Table shows a summary of the results of the DOEanalysis indicating the statistically significant responses andassociated terms. The responses considered in the DOE analysis were therate of change per day in HMW, LMW, and monomer by SE-UPLC, and the rateof change per day in acidic species, main peak, and basic species byCEX-UPLC and icIEF. Other responses, including visual appearance,turbidity by OD 405 nm, pH, protein concentration (recovery) andsub-visible particles by MFI, were measured but were consideredpass/fail or for information and not used in the DOE analysis.

TABLE 23 Statistically significant responses, associated factors, ratesand P-values. Impact to the rate Response Term per day^(a) p-value^(b)Acidic rate 45° C. per day pH 0.116 0.000 Main rate 45° C. per day pH−0.079 0.000 Basic rate 45° C. per day pH −0.037 0.000 icIEF basic 45°C. per day pH −0.025 0.002 Acidic rate 45° C. per day Histidine*pH 0.0150.003 Monomer rate 45° C. per day Sucrose 0.005 0.003 HMW rate 45° C.per day garetosmab 0.004 0.004 HMW rate 45° C. per day Sucrose −0.0040.004 Monomer rate 45° C. per day garetosmab −0.004 0.004 HMW rate 45°C. per day pH −0.004 0.004 Monomer rate 45° C. per day pH 0.004 0.009Acidic rate 45° C. per day garetosmab*pH −0.010 0.011 icIEF acidic 45°C. per day pH 0.055 0.018 icIEF basic 45° C. per day Histidine*Histidine−0.033 0.025 Acidic rate 45° C. per day Arginine −0.008 0.031 Monomerrate 45° C. per day Arginine −0.003 0.037 icIEF basic 45° C. per dayArginine*Arginine 0.026 0.041 Highlighted in bold are the statisticallysignificant, non-main effect terms based on stress stability testing at45° C. ^(a)Rates are derived from scaled estimates reported in JMP. Thereported rates are 2-times the scaled estimates, as the scaled estimatesare half-estimates. ^(b)p-values ≤ 0.05 are considered significant inthis study.

Results of visual inspection, turbidity by OD 405 nm, pH, proteinrecovery and sub-visible particles by MFI were: all samples passedvisual inspection; no meaningful increases in OD 405 nm were observedfor any sample; no changes in protein concentrations were observed. Allconcentration measurements were within 5% of the t=0 values; and somefluctuations in particle concentrations by MFI were observed as well assome increasing trends. Some trends were statistically significant butthose that were significant were not practically meaningful.

Conclusions

The results of the DOE analysis indicated that the following non-maineffect factors had a statistically significant effect on garetosmabstability under thermal stress conditions (incubation at 45° C. for 28days).

-   -   [Histidine]*pH    -   [garetosmab]*pH    -   [Histidine]*[Histidine]    -   [Arginine]*[Arginine]

These interactions and quadratics were included in the final robustnessstudy along with all main effects. A detailed summary of the preliminaryrobustness study including data from incubation at 45° C. and 25° C. isdiscussed in report EXP-9 Jul. 2019-0096 garetosmab Pre-PAR Study.

Garetosmab Final Robustness Study Design

The formulation factor ranges tested in this study were defined to bewider than or equal to the ranges that were anticipated to occur if oneconsiders both contributions from manufacturing variability and assayvariability. The formulation parameters in this robustness study includegaretosmab concentration (±10% which is the specification limit), bufferand stabilizer concentrations (±20%), surfactant concentration (±50%),and pH (±0.3 units which is the specification limit) (Table).

To evaluate the impact of the key formulation factors identified fromthe preliminary robustness study on the storage and stress stability ofthe garetosmab formulation, a 6-factor D-optimal DOE design was executedto characterize and explore the excipient design space. A 14-run study(Table) was designed which considered all main effects, and thesecondary interactions and quadratics identified as statisticallysignificant in the preliminary robustness study. The design was created,evaluated and confirmed in JMP12.1 to be sufficiently powered and ableto cover the design space with good estimation capability. In additionto the test formulations, one formulation with all factors at thenominal levels was included as a control but excluded from the DOEanalysis. 10 mL type 1 glass vials were filled with 5.5 mL offormulation, which is representative of the commercial DP presentation.Each formulation was assessed for long-term storage stability at 2° C.to 8° C., accelerated stability at 25° C./60% RH and stress conditions;40° C./75% RH, and agitation and freezing and thawing stress (storageand stress conditions are summarized in Table 26: Analysis Plan forgaretosmab

Assay Time Points Purpose Visual appearance All Color change,precipitation, particles Turbidity, OD @ 405 nm Precipitation pH pHchanges RP-HPLC Protein concentration and recovery SE-HPLC Chemicalstability, high and low MW species CEX-HPLC Chemical stability, chargevariants, deamidation icIEF 0, 3, 6, 12, 18, 24, 36, Chemical stability,charge variants, 48 and 60 month 5° C. deamidation MCE-SDS (R and NR)120 minutes agitation Chemical stability, high and low MW species MFI 8xfreeze/thaw Particles HIAC 1, 2 months Particles 40° C./75% RH 3, 6months 25° C./60% RH CAD-UPLC 0, 3, 6, 12, 24, 36, 48 Polysorbate 20Concentration Bio-Assay and 60 month 5° C. Potency/Biological activity

).

All formulations were characterized and assessed for physical andchemical properties and stability, including visual inspection, pH,turbidity, protein concentration and recovery, purity with respect tomolecular weight forms (assessed by SE-UPLC and microchip capillaryelectrophoresis (MCE)), and charge variants (assessed by CEX-UPLC andimage capillary iso-electric focusing (icIEF)), and subvisibleparticulate analysis (assessed by light obscuration (HIAC) and MFI), assummarized in Table 26.

TABLE 24 Formulations assessed in the garetosmab final robustness studyProtein, Histidine, Sucrose, Arginine, Polysorbate Formulation # Lot #mg/mL mM pH % (w/y) mM 20, % (w/v) F1 L18-3972 66 8 6 4 56 0.025 F2L18-3973 54 12 6 6 56 0.025 F3 L18-3974 66 8 6.6 6 70 0.075 F4 L18-397566 10 6 6 84 0.075 F5 L18-3976 54 8 6.6 4 84 0.025 F6 L18-3977 66 12 6 456 0.075 F7 L18-3978 54 10 6 4 70 0.025 F8 L18-3979 54 10 6.6 6 56 0.025F9 L18-3980 54 12 6.6 4 70 0.075 F10 L18-3981 54 8 6 6 56 0.075 F11L18-3982 66 8 6 6 70 0.025 F12 L18-3983 66 10 6.6 4 56 0.075 F13L18-3984 54 8 6 4 84 0.075 F14 L18-3985 66 12 6.6 6 84 0.025 F15^(a)L18-3986 60 10 6.3 5 70 0.050 ^(a)Formulation 15 was a controlformulation where all components were included at the nominal levels ofthe commercial formulation. This formulation was not included in any ofthe DOE analyses but was used as a relative measure of the stability ofthe test formulations.

TABLE 25 Storage conditions and sampling intervals for garetosmab finalrobustness study Storage/Stress Conditions Sampling Intervals Agitation(vortex) 30, 60 and 120 minutes Freeze/Thaw (−30° C.) 4x and 8x 5° C. 1,3, 6, 9, 12, 18, 24, 36, 48, 60 months 25° C./60% RH 1, 2, 3, 6 months40° C./75% RH 0.5, 1 and 2 months T = 0 0 (days)

TABLE 26 Analysis Plan for garetosmab Assay Time Points Purpose Visualappearance All Color change, precipitation, particles Turbidity, OD @405 nm Precipitation pH pH changes RP-HPLC Protein concentration andrecovery SE-HPLC Chemical stability, high and low MW species CEX-HPLCChemical stability, charge variants, deamidation icIEF 0, 3, 6, 12, 18,24, 36, Chemical stability, charge variants, 48 and 60 month 5° C.deamidation MCE-SDS (R and NR) Chemical stability, high and low MWspecies MFI 120 minutes agitation Particles HIAC 8x freeze/thawParticles 1, 2 months 40° C./75% RH 3, 6 months 25° C./60% RH CAD-UPLC0, 3, 6, 12, 24, 36, 48 Polysorbate 20 Concentration Bio-Assay and 60month 5° C. Potency/Biological activity

Results (Stress Temperature 40° C./75% RH)

Garetosmab samples in the formulations listed in Table 24 were incubatedat 40° C./75% RH for 2 months. The responses that were considered mostimportant for the DOE analysis were formation of molecular weightvariants by SE-UPLC and MCE, and formation of charge variants byCEX-UPLC and icIEF. Additional factors including visual inspection, pH,turbidity, protein concentration and recovery, and particulate levelswere measured but were considered pass/fail or for information and notused in the DOE analysis. The factors that led to statisticallysignificant changes in the measured responses are listed in Table.Although several factors had statistically significant impacts to theresponses, the effects were small, resulting in relatively small changesin response to variations in the factors under this stress condition.The factor that had the largest effect was pH on the rate of chargevariant formation by CEX-UPLC. Although statistically significant, theeffect was small. The rate of formation of acidic species varied by 3.8%per month when the pH varied from 6.0-6.6. Overall, under the stresscondition tested, the variations in the formulation had little impact tothe stability of garetosmab. The variation in the rates is illustratedin FIG. 2. In all cases the spread relative to the control is notmeaningful with respect to the overall stability of the formulation.

TABLE 27 Statistically significant responses from the final robustnessstudy for garetosmab formulations incubated at 40° C./75% RH for 2months. Impact to the rate Response^(a) Factor per month^(b) p-value^(c)HMW garetosmab 0.058 0.0011 HMW pH −0.052 0.0022 HMW Sucrose −0.0400.0081 HMW Polysorbate 20 0.028 0.0357 Acidic CEX pH 3.771 <0.0001Acidic CEX Arginine −0.459 0.0230 Acidic CEX Histidine*pH 0.850 0.0008Main Peak CEX pH 2.492 <0.0001 Basic CEX pH −1.250 <.0001 Basic CEXHistidine*pH −0.654 0.0145 Acidic icIEF pH 2.334 <0.0001 Acidic icIEFHistidine*pH 1.146 0.0042 Main Peak icIEF garetosmab 0.476 0.0374 MainPeak icIEF pH 1.804 <0.0001 Main Peak icIEF Histidine*pH 0.783 0.0102Basic icIEF garetosmab 0.244 0.0429 Basic icIEF pH −0.524 0.0014 BasicicIEF Arginine 0.311 0.0376 Basic icIEF Histidine*pH −0.344 0.0251Purity reduced MCE garetosmab −0.333 0.0245 Purity reduced MCE pH −0.3690.0159 Purity reduced MCE Sucrose 0.379 0.0137 LMW reduced MCEgaretosmab 0.246 0.0465 LMW reduced MCE pH 0.343 0.0108 ^(a)Reported asrate of formation or loss of the species monitored. ^(b)Rates arederived from scaled estimates reported in JMP. The reported rates are2-times the scaled estimates, as the scaled estimates arehalf-estimates. ^(c)p-values ≤ 0.05 were considered statisticallysignificant.

Results (Accelerated Temperature 25° C./60% RH)

Garetosmab samples in the formulations listed in Table were incubated at25° C./60% RH for up to 6 months. The responses that were consideredmost important for the DOE analysis were formation of molecular weightvariants by SE-UPLC and MCE, and formation of charge variants byCEX-UPLC and icIEF. Additional factors including visual inspection, pH,turbidity, protein concentration and recovery, and particulate levelswere measured but were considered pass/fail or for information and notused in the DOE analysis. The factors that led to significant changes inthe measured responses are listed in Table. Although several factors hadstatistically significant impacts to the responses, the effects weresmall, resulting in less than a 1% variation in the rate per month dueto varying the factors, under this accelerated condition. Under theaccelerated condition tested, the variations in the formulation hadlittle impact to the stability of garetosmab. The variation in the ratesis illustrated in FIG. . In all cases the spread relative to the controlis not meaningful with respect to the overall stability of theformulation.

TABLE 28 Statistically significant responses from the final robustnessstudy for garetosmab formulations incubated at 25° C./60% RH for 6months. Impact to the rate Response^(a) Factor per month^(b) p-value^(c)HMW garetosmab 0.007 0.0011 HMW pH 0.004 0.0212 HMW Sucrose −0.0050.0067 Acidic CEX Histidine 0.089 0.0415 Acidic CEX pH 0.730 <0.0001Acidic CEX Histidine*pH 0.120 0.0103 Main Peak CEX pH 0.526 <0.0001Basic CEX Histidine −0.040 0.0272 Basic CEX pH −0.210 <0.0001 Basic CEXHistidine*pH −0.052 0.0096 Acidic icIEF Histidine 0.198 0.0267 AcidicicIEF pH 0.577 <0.0001 Acidic icIEF garetosmab*pH −0.162 0.0283 AcidicicIEF Histidine*pH 0.404 0.0007 Main Peak icIEF Histidine 0.169 0.0255Main Peak icIEF pH 0.446 <0.0001 Main Peak icIEF garetosmab*pH −0.1210.0458 Main Peak icIEF Histidine*pH 0.247 0.0044 Basic icIEF pH −0.1390.0064 Basic icIEF Histidine*pH −0.157 0.0079 Purity reduced MCE pH−0.092 0.0142 Purity reduced MCE garetosmab*pH −0.080 0.0298 LMW reducedMCE garetosmab 0.042 0.0191 LMW reduced MCE pH 0.067 0.0034 LMW reducedMCE garetosmab*pH 0.042 0.0191 LMW reduced MCE Histidine*pH −0.0810.0029 LMW reduced MCE Histidine*Histidine −0.106 0.0112 LMW non-reducedMCE Histidine*Histidine 0.142 0.0396 LMW non-reduced MCEArginine*Arginine 0.153 0.0313 ^(a)Reported as rate of formation or lossof the species monitored. ^(b)Rates are derived from scaled estimatesreported in JMP. The reported rates are 2-times the scaled estimates, asthe scaled estimates are half-estimates. ^(c)p-values ≤ 0.05 wereconsidered statistically significant.

Results (Stress Conditions Agitation and Freezing and Thawing)

The stability of the garetosmab formulations listed in Table 24 wereassessed after 4 or 8 cycles of freezing and thawing, or after 30, 60 or120 minutes of agitation by vortexing. The quality attributes assessedwere formation of molecular weight variants by SE-UPLC and MCE,formation of charge variants by CEX-UPLC and icIEF, visual inspection,pH, turbidity, protein concentration and recovery, and particulatelevels. FIG. and FIG. show summaries of the impacts of varying thecomponents of the formulation, in accordance with the DOE design,relative to the control formulation where all components of theformulation were nominal, after 8 cycles of freezing and thawing or 120minutes of agitation by vortexing (indicated on the graphs as “deltacontrol”). These results indicate that varying the formulationcomposition has no meaningful impact to the stability of garetosmab,relative to the control formulation, under the stress conditionsemployed.

Results (Long-Term Storage Temperature 2-8° C.)

Garetosmab samples, prepared in accordance with the DOE design, in theformulations listed in Table 24 were stored at 2-8° C. Currently, 24months of long-term stability data for the garetosmab robustness studyare available. The effects of varying garetosmab concentration, pH,histidine concentration, arginine concentration, sucrose concentrationand polysorbate 20 concentration on the long-term stability ofgaretosmab were examined. The quality attributes assessed were formationof molecular weight variants by SE-UPLC and MCE, formation of chargevariants by CEX-UPLC and icIEF, visual inspection, pH, turbidity,protein concentration and recovery, and particulate levels. FIG. 7 showa summary of the impact of varying the components of the formulation, inaccordance with the DOE design, relative to the control formulationwhere all components of the formulation were nominal. These resultsindicate that after 12 or 24 months of storage at 5° C., varying theformulations within the ranges tested resulted in no meaningful impactto the stability or quality of garetosmab. No precipitate or visibleparticulate was detected by either visual inspection or turbiditymeasurements (OD at 405 nm). No meaningful changes in protein recoverywere observed (RP-UPLC). The pH of the formulations was stable. Noappreciable increases in subvisible particulates were observed by HIAC,and no appreciable differences were observed in the subvisibleparticulate counts relative to the control formulation: for subvisibleparticulates measured by HIAC, all values were below the acceptablelimits set by USP <788>; subvisible particles were also measured by MFI.Particle counts were variable but no meaningful changes in subvisibleparticles were observed relative to the control or between formulationsfor 10 μm or 25 μm particles. A trend of increasing 2-10 μm particlecounts is observed with time, but the differences are not consideredappreciable.

Statistical analysis of the results indicate that the differences amongthe formulations is primarily due to random variation and not due tovariation in the factors (FIGS. 6-7); relative to the controlformulation, no meaningful differences were observed in the levels ofHMW species, LMW species or Native garetosmab as determined by SE-UPLCor MCE; and relative to the control formulation, no meaningfuldifferences were observed in the levels of acidic or basic chargevariants as determined by CEX-UPLC or icIEF.

The results from this study demonstrated that varying the formulationfactors (garetosmab concentration, pH, histidine concentration, arginineconcentration, sucrose concentration and polysorbate 20 concentration)within the studied ranges have no appreciable impact on the garetosmablong-term stability. The 60 mg/mL garetosmab formulation is robust withrespect to all the quality attributes within the tested formulationcomposition range.

Garetosmab Formulation Robustness Summary

Formulation robustness studies were conducted to evaluate the effect ofvariations in formulation parameters on the garetosmab formulationstability. A DOE study that evaluated long-term storage, accelerated andstress stability demonstrated that variation in the formulationparameters, within the range studied, did not appreciably affectgaretosmab quality and stability.

Specifically, the 60 mg/mL proposed commercial garetosmab formulation isrobust against a ±10% variation in protein concentration, a ±20%variation in sucrose, arginine and/or histidine concentration, a ±50%variation in polysorbate 20 concentration, and/or a ±0.3 pH unitvariation. Overall, the results from the 60 mg/mL garetosmab robustnessstudy support that the variation in the composition of the garetosmabformulation within the ranges studied will not adversely impact thestability or quality of the garetosmab DP under the recommended storageconditions (2-8° C.).

Conclusions

Based on the results from the formulation development studies that werepresented, as well as the clinical experience, the commercial garetosmabformulation for IV infusion was developed, which contains the followingcomponents: 60 mg/mL Garetosmab, 10 mM L-histidine, 5% (w/v) sucrose, 70mM L-arginine HCl, 0.05% (w/v) polysorbate 20 and pH 6.3. Thiscommercial IV formulation meets the goals defined for formulationdevelopment. The developed formulation is a liquid formulation in glassvials with a concentration of garetosmab sufficient to deliver a dose of10 mg/kg garetosmab by intravenous (IV) infusion. The garetosmab DP is60 mg/mL and is manufactured with a 5 mL withdrawable volume providing300 mg per vial. The formulation is a near iso-osmolar formulation thatis compatible with 0.9% Sodium Chloride Injection or 5% DextroseInjection for IV infusion. The formulation is compatible with and stablein type 1 clear glass vials and standard serum stopper as the primarypackaging system. The formulation is a sterile DP that supportslong-term stability with a shelf-life of 24 months or longer at 2-8° C.No meaningful changes were observed in any garetosmab quality attributeswhen stored at 2-8° C. for up to 12 months, 18 months or 24 months. Theformulation is a robust formulation that minimizes formation ofgaretosmab high molecular weight (HMW) species, minimizes changes in therelative distribution of garetosmab charge variant species, maintainssubvisible particles at a safe level, and maintains the biologicalactivity when subjected to handling and thermal stresses. Theformulation is a formulation that is easily converted for use insubcutaneous (SC) administration. The garetosmab formulated drugsubstance is 200 mg/mL.

Example 5: Overages

There are no overages included in the formula; however, a slightoverfill was included to compensate for the normal variations in fillvolume encountered during an automated fill finish process and to ensurethat the correct volume can be withdrawn from the vial. Clinical DP wasmanufactured with a 5.44 mL minimum fill volume. An overfill of 0.44 mLwas contained in each vial, which is sufficient for an accuratewithdrawal of 5.0 mL (300 mg) of DP from the vial.

The overage was not designed to compensate for losses duringmanufacture, degradation during manufacture, degradation during storage(shelf life), or to extend the expiration dating period. These overageswere intended to enable the accurate withdrawal and administration of5.0 mL DP (e.g., 300 mg of garetosmab).

Example 6: Methods Used to Assess Stability

The research stability of garetosmab DP was assessed using differentassays. Color and appearance were assessed by visual inspection.Turbidity measured by increase in optical density (OD) at 405 nm.Particulate matter analysis was determinded by light obscuration (HIAC).Particulate matter analysis was assessed by Micro-Flow Imaging™ (MFI).Protein concentration was assessed by reversed-phase ultra-performanceliquid chromatography (RP-UPLC). Purity of each individual DP wasassessed by using the following assays: size-exclusion ultra-performanceliquid chromatography (SE-UPLC); reduced and non-reduced microchipcapillary electrophoresis (MCE).

Charge variant analysis was determined using the following assays:Cation exchange UPLC (CEX-UPLC); and imaged capillary isoelectricfocusing (iCIEF). Charge variants are reported as percent Region 1,Region 2, and Region 3. Region 1 corresponds to acidic species whichelute before the Main Peak, Region 2 corresponds to the Main Peak, andRegion 3 corresponds to basic species which elute after the Main Peak.

The Potency was assessed by use of a bioassay. The relative potency ofeach sample was determined by bioassay and is defined as: (IC₅₀reference sample/IC₅₀ test sample)×100%. The measured potency of storagestability samples must be within 50-150% of the measured potency of thereference standard.

INFORMAL SEQUENCE LISTING SEQ ID NO.: 1  GGSFSSHF  SEQ ID NO.: 2 ILYTGGT  SEQ ID NO.: 3  ARARSGITFTGIIVPGSFDI  SEQ ID NO.: 4  QSVSSSY SEQ ID NO.: 5  GAS  SEQ ID NO.: 6  QQYGSSPWT  SEQ ID NO: 7 QVQLQESGPG LVKPSETLSL TCTVSGGSFS SHFWSWIRQPPGKGLEWIGY ILYTGGTSFN PSLKSRVSMS VGTSKNQFSLKLSSVTAADT AVYYCARARS GITFTGIIVP GSFDIWGQGT MVTVSS  SEQ ID NO: 8 EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQKPGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLEPEDFAVYYCQ QYGSSPWTFG QGTKVEIK 

What is claimed is:
 1. A pharmaceutical formulation comprising: (i) ananti-human Activin A antibody, or antigen-binding portion thereof; (ii)a buffer at pH of 6.3±0.3; (iii) an organic cosolvent; and (iv) one ormore thermal stabilizers.
 2. The pharmaceutical formulation of claim 1,wherein the antibody, or the antigen-binding portion thereof, comprisesthe following six CDR sequences: (a) an HCDR1 having the sequenceGGSFSSHF (SEQ ID NO.: 1); (b) an HCDR2 having the sequence ILYTGGT (SEQID NO.: 2); (c) an HCDR3 having the sequence ARARSGITFTGIIVPGSFDI (SEQID NO.: 3); (d) an LCDR1 having the sequence QSVSSSY (SEQ ID NO.: 4);(e) an LCDR2 having the sequence GAS (SEQ ID NO.: 5); and (f) an LCDR3having the sequence QQYGSSPWT (SEQ ID NO.: 6).
 3. The pharmaceuticalformulation of claim 2, wherein the antibody has a molecular weight ofabout 145,235.3 Da.
 4. The pharmaceutical formulation of claim 3,wherein the antibody, or the antigen-binding portion thereof,concentration is 60 mg/mL±6 mg/mL.
 5. The pharmaceutical formulation ofclaim 1, wherein the buffer is a histidine buffer.
 6. The pharmaceuticalformulation of claim 5, wherein the histidine concentration is 10 mM±2mM.
 7. The pharmaceutical formulation of claim 1, wherein the organiccosolvent is polysorbate
 20. 8. The pharmaceutical formulation of claim7, wherein the polysorbate 20 concentration is 0.05% w/v±0.025%.
 9. Thepharmaceutical formulation of claim 1, wherein the one or more thermalstablilizers comprise sucrose and arginine.
 10. The pharmaceuticalformulation of claim 9, wherein the sucrose concentration is 5%±1% (w/v)and the Arginine concentration is 70 mM±14 mM.
 11. The pharmaceuticalformulation of claim 1, comprising 60 mg/mL±6 mg/mL antibody, 10 mM±2 mMhistidine, pH 6.3±0.3, 0.05% w/v±0.025% polysorbate 20, 5% w/v±1%sucrose, and 70 mM±14 mM Arginine.
 12. The pharmaceutical formulation ofclaim 2, wherein after 56 days of storage at 40° C. and 75% relativehumidity (RH), at least 90% of the antibody, or the antigen-bindingportion thereof, has native conformation, or at least 30% of theantibody, or the antigen-binding portion thereof, is the main chargeform.
 13. The pharmaceutical formulation of claim 2, wherein after 56days of storage at 40° C. and 75% RH, at least 93% of the antibody, orthe antigen-binding portion thereof, has native conformation, or atleast 34.5% of the antibody, or the antigen-binding portion thereof, isthe main charge form.
 14. The pharmaceutical formulation of claim 2,wherein after 56 days of storage at 40° C. and 75% RH, at least 97% ofthe antibody, or the antigen-binding portion thereof, has nativeconformation, or at least 45% of the antibody, or the antigen-bindingportion thereof, is the main charge form.
 15. The pharmaceuticalformulation of claim 2, wherein after six months of storage at 25° C.and 60% RH, at least 90% of the antibody, or the antigen-binding portionthereof, has native conformation, or at least 40% of the antibody, orthe antigen-binding portion thereof, is the main charge form.
 16. Thepharmaceutical formulation of claim 2, wherein after six months ofstorage at 25° C. and 60% RH, at least 95% of the antibody, or theantigen-binding portion thereof, has native conformation, or at least45% of the antibody, or the antigen-binding portion thereof, is the maincharge form.
 17. The pharmaceutical formulation of claim 2, whereinafter six months of storage at 25° C. and 60% RH, at least 98% of theantibody, or the antigen-binding portion thereof, has nativeconformation, or at least 50% of the antibody, or the antigen-bindingportion thereof, is the main charge form.
 18. The pharmaceuticalformulation claim 2, wherein after 12 months of storage at 2-8° C., atleast 94% of the antibody, or the antigen-binding portion thereof, hasnative conformation, at least 45% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 100% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 19. Thepharmaceutical formulation claim 2, wherein after 12 months of storageat 2-8° C., at least 96% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 50% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 100% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 20. Thepharmaceutical formulation claim 2, wherein after 12 months of storageat 2-8° C., at least 98% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 55% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 100% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 21. Thepharmaceutical formulation claim 2, wherein after 18 months of storageat 2-8° C., at least 94% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 45% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 95% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 22. Thepharmaceutical formulation claim 2, wherein after 18 months of storageat 2-8° C., at least 96% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 50% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 95% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 23. Thepharmaceutical formulation claim 2, wherein after 18 months of storageat 2-8° C., at least 98% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 55% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 95% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 24. Thepharmaceutical formulation claim 2, wherein after 24 months of storageat 2-8° C., at least 94% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 45% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 99% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 25. Thepharmaceutical formulation claim 2, wherein after 24 months of storageat 2-8° C., at least 96% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 50% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 99% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 26. Thepharmaceutical formulation claim 2, wherein after 24 months of storageat 2-8° C., at least 98% of the antibody, or the antigen-binding portionthereof, has native conformation, at least 55% of the antibody, or theantigen-binding portion thereof, is the main charge variant, and/or theantibody retains at least 99% of the potency of the antibody, or theantigen-binding portion thereof, prior to storage.
 27. A pharmaceuticalformulation comprising (a) 60 mg/mL±10 mg/mL of an anti-human Activin Aantibody, or antigen-binding portion thereof (b) 10 mM±2 mM histidine,pH 6.3±0.3, (c) 0.05%±0.025% polysorbate 20, (d) 70 mM±14 mM Arginine,and (e) 5%±1% sucrose, wherein the antibody, or the antigen-bindingportion thereof, comprises an heavy chain variable region comprising SEQID NO: 7 and a light chain variable region comprising SEQ ID NO:
 8. 28.The pharmaceutical formulation of claim 1, wherein the formulation iscontained in a container.
 29. The pharmaceutical formulation of claim28, wherein the container is a vial.
 30. The pharmaceutical formulationof claim 29, wherein the vial is glass.
 31. The pharmaceuticalformulation of claim 30, wherein the glass is Type 1 borosilicate glasswith a FluroTec® coated 4432/50 butyl rubber stopper.
 32. Thepharmaceutical formulation of claim 1, wherein the formulation issuitable for intravenous administration to a human subject in needthereof.
 33. The pharmaceutical formulation of claim 1, wherein theformulation is suitable for subcutaneous administration to a humansubject in need thereof.
 34. The pharmaceutical formulation of claim 1,wherein the formulation is a liquid formulation.
 35. The pharmaceuticalformulation of claim 1, wherein the formulation is a lyophilizedformulation.
 36. A kit comprising a pharmaceutical formulation of claim1, a container, and instructions of use thereof.
 37. The kit of claim36, wherein the container is a glass vial fitted with a FluroTec® coatedchlorobutyl stopper.
 38. A method of treating a disease or disorderassociated with Activin A activity, the method comprising administrationof a therapeutically effective amount of the pharmaceutical compositionof claim 1 to a subject in need thereof.
 39. The method of claim 38,wherein the disease or disorder associated with Activin A activity isFibrodysplasia ossificans progressiva (FOP).